( C ) Relative protein abundance of EGFP - Abeta42 ( A42 ) versus empty vector ( ev ) expressing wild type yeast cells . The significant subset of cytosolic heat shock proteins ( HSP ) detected in a proteomics analysis of isolated mitochondria is depicted . Log2 SILAC ratios of A42 / ev of two independent proteome measurements are shown . Dot plots show all data points along with the mean ( line ) . Significance was determined using an outlier test ( Significance A , p < 0 . 003 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Relative protein abundance of EGFP-Abeta42 (A42) versus empty vector (ev) expressing wild type yeast cells. The significant subset of cytosolic heat shock proteins (HSP) detected in a proteomics analysis of isolated mitochondria is depicted. Log2 SILAC ratios of A42/ev of two independent proteome measurements are shown. Dot plots show all data points along with the mean (line). Significance was determined using an outlier test (Significance A, p <0.003).


( D ) Screen of 123 deletion strains assessing Abeta42 ( A42 ) - induced toxicity ( assessed by DHE > Eth . positive cells indicative of the sum of sub - lethal oxidative stress and cell death ) in yeast . Relative A42 - toxicity normalized to wild type ( WT ) cells is depicted . Potential hits ( mutants that reduce A42 toxicity ) are shown in green . Data represent means ± s . d . . n ≥ 3 biologically independent cultures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Screen of 123 deletion strains assessing Abeta42 (A42)-induced toxicity (assessed by DHE>Eth. positive cells indicative of the sum of sub-lethal oxidative stress and cell death) in yeast. Relative A42-toxicity normalized to wild type (WT) cells is depicted. Potential hits (mutants that reduce A42 toxicity) are shown in green. Data represent means ± s.d.. n ≥ 3 biologically independent cultures.


Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) of wild type ( WT ) ( A ) and YDJ1 deleted ( Δydj1 ) cells ( B ) . Mean ± s . d . . n = 5 biologically independent cultures . Comparisons by two - way ANOVA ( mixed - design ) followed by simple main effects ( * * * , p < 0 . 001 , vs . control ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP-A42 (A42) or EGFP empty vector (ev) of wild type (WT) (A) and YDJ1 deleted (Δydj1) cells (B). Mean ± s.d.. n = 5 biologically independent cultures. Comparisons by two-way ANOVA (mixed-design) followed by simple main effects (***, p < 0.001, vs. control).


Immunoblot of whole cell extract ( Input ) and eluate of FLAG - tagged Ydj1 . Immunoprecipitation ( IP : FLAG ) of YDJ1 deletion strain ( Δydj1 ) cells expressing EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) and co - overexpressing Ydj1 - FLAG using Abeta - specific antibody ( 6E10 ) and FLAG antibody ( FLAG ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot of whole cell extract (Input) and eluate of FLAG-tagged Ydj1. Immunoprecipitation (IP: FLAG) of YDJ1 deletion strain (Δydj1) cells expressing EGFP-A42 (A42) or EGFP empty vector (ev) and co-overexpressing Ydj1-FLAG using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).


Confocal microscopy of wild type ( WT ) yeast cells expressing EGFP - A42 ( A42 ) only and co - overexpressing Ydj1 - FLAG or with the corresponding vector control after 18 h of expression . Colors indicate fluorescence intensity .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Confocal microscopy of wild type (WT) yeast cells expressing EGFP-A42 (A42) only and co-overexpressing Ydj1-FLAG or with the corresponding vector control after 18 h of expression. Colors indicate fluorescence intensity.


Confocal microscopy of wild type ( WT ) yeast cells expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG after 18 h of expression . Mitochondria were visualized with MitoTracker Red ( magenta ) . See also Fig EV2J , EV1C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal microscopy of wild type (WT) yeast cells expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG after 18 h of expression. Mitochondria were visualized with MitoTracker Red (magenta). See also Fig EV2J, EV1C.


Immunoblot of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) and cytosolic ( Cyt . ) fractions of wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) cells after 18 h of expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time exposure ( Short exp . ) . Tom22 - specific antibody is a marker for mitochondria , Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.) and cytosolic (Cyt.) fractions of wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Tom22-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.


Ratio of full length EGFP - A42 in the mitochondrial fraction / cytosolic fraction , using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three / 4 independent experiments . Dot plots show all data points along with the mean ( line / bar ) ± s . d . . n = 3 - 4 biologically independent cultures . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ratio of full length EGFP-A42 in the mitochondrial fraction / cytosolic fraction, using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three/4 independent experiments. Dot plots show all data points along with the mean (line/bar) ± s.d.. n = 3-4 biologically independent cultures. *, p < 0.05. Unpaired, two-tailed t-test.


Assay for protein degradation using cycloheximide ( CH ) to stall protein translation . EGFP fluorescence intensity was measured at two time points ( t0 and t2 , 2 h after CH administration ) and normalized to t0 in wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) cells after 18 h of expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc . See also Appendix Fig S2D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity was measured at two time points (t0 and t2, 2 h after CH administration) and normalized to t0 in wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 8 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc. See also Appendix Fig S2D.


Quantification of the ratio between EGFP - A42 tetramer and monomer in wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) expressing EGFP - A42 ( J ) as well as between WT expressing EGFP - A42 only and co - overexpressing Ydj1 - FLAG ( K ) at indicated time points . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 ( J ) n = 12 ( K ) biologically independent cultures . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc test . See also Appendix Fig S2A - B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the ratio between EGFP-A42 tetramer and monomer in wild type (WT) and YDJ1 deletion strain (Δydj1) expressing EGFP-A42 (J) as well as between WT expressing EGFP-A42 only and co-overexpressing Ydj1-FLAG (K) at indicated time points. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8(J) n=12(K) biologically independent cultures. ***, p < 0.001; **, p < 0.01; *, p < 0.05. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2A-B.


Quantification of propidium iodide ( PI ) - staining positive cells indicating cell death at indicated time points of expression of EGFP - A42 ( A42 ) Δydj1 cells with co - expression of DnaJA1 - FLAG ( DnaJA1 ) ( B ) or corresponding empty vector controls ( ev ) ( A ) . Mean ± SD n = 4 - 6 biologically independent cultures . Comparisons by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus control ) .

Quantification of propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4-6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control).


( C ) Immunoblot ( WB ) of whole - cell extract ( Input ) and eluate of FLAG - tagged DnaJA1 immunoprecipitation ( IP : FLAG ) of wild - type strain ( WT ) expressing EGFP - A42 ( A42 ) and co - expressing DnaJA1 - FLAG ( A1 ) using Abeta - specific antibody ( 6E10 ) and FLAG antibody ( FLAG ) .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).


Assay for protein degradation using cycloheximide ( CH ) to stall protein translation . EGFP fluorescence intensity of YDJ1 deletion strain ( Δydj1 ) and wild type ( WT ) cells expressing EGFP - A42 ( A42 ) only or co - expressing DnaJA1 was measured at two time points ( t0 and t2 ) and normalized to t0 . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 6 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc test . See also Appendix Fig S2C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C.


Immunoblot ( WB ) of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) , and cytosolic ( Cyt . ) fractions of YDJ1 - deleted cells ( Δydj1 ) after 18 h expression of EGFP - A42 ( A42 ) only or co - expressing DnaJA1 - FLAG ( DnaJA1 ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time ( Short exp . ) exposure ( two sections from one immunoblot ) . Cox4 - specific antibody is a marker for mitochondria , Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.


Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 ( A1 ) and quantified by densitometry in ( G ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G).


Quantification of the oligomer - to - monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in ( F ) . Dot plots show all data points along with the mean ( line ) ± SD n = 3 biologically independent cultures . P - values by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus A42 control ) . See also Appendix Fig S2D .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D.


A42 beta - sheet - rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ . Data represent means of at least eight measurements . See also Appendix Fig S1E .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.


( A ) Immunoblot of cytosol - enriched cerebral tissue homogenate ( Input ) and eluate of Abeta42 or DnaJA1 using Abeta - specific antibody ( 6E10 ) and DnaJA1 - specific antibody ( DnaJA1 ) , respectively , showing optimal and long exposure ( Long exp . ) . Immunoprecipitation using magnetic beads without antibody , DnaJA1 or 6E10 antibody of female ( 15 months old ) wild type ( WT ) or 3xTg ( PS1M146V / APPSwe / tauP301L ) mice . Sections showing monomer and low - n oligomers , dodecamer and full - length amyloid precursor protein ( APP ) are from 1 immunoblot .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.


( B ) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients ( AD ) and aged non - demented controls ( ctrl ) . PonceauS serves as a loading control . ( C ) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients ( AD ) and aged non - demented controls ( ctrl ) . Representative immunoblot is shown in ( B ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 4 - 5 independent patients . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control. (C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.


( A ) Representative confocal microscopy of 10 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and reference nuclei staining with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( B ) Representative confocal and gSTED deconvolved ( decon ) images of Kenyon cells in 18 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and reference nuclei staining with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( C - D ) Average intensity ( C ) and total area ( D ) of Abeta ( 6E10 ) signal from confocal images representatively shown in ( A ) from 12 brains of w1118 wild type ( Droj2 + / + ) and knockdown ( Droj2 + / - ) 10 - day - old male flies . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 12 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 10-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 12. *, p < 0.05. Unpaired, two-tailed t-test.


( E - F ) qPCR analysis of Droj2 - mRNA levels of 3 - 6 days old female ( E ) and male ( F ) flies expressing human Abeta42 ( UAS - A42 ) of w1118 wild type flies ( Droj2 + / + ) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression ( Droj2 + / + ctrl ) . Reference gene is Rpl32 . Dot plots show all data points along with the mean ( line ) ± s . d . . n = 3 . * * , p < 0 . 01 . One sample t - test against 1 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.


( G ) Immunoprecipitation ( IP : 6E10 ) of w1118 wild type flies ( Droj2 + / + ) using synthetic Abeta42 added to the fly head extract ( Input ) . Abeta - specific antibody 6E10 was used for Abeta42 and DnaJA1 - specific antibody for Droj2 immunoblot detection . Showing input , supernatant , supernatant after washing ( WS ) and eluate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate.


( A - B ) Survival of female ( A ) and male ( B ) w1118 wild type flies ( Droj2 + / + ) and Droj2 knockdown flies ( Droj2 + / - ) with expression of human Abeta42 ( UAS - A42 ) or control flies without expression ( ctrl ) , upon supplementation of sugar ( 10 % sucrose ) with 20 mM MnCl2 . Survival was determined at indicated time points . n = 6 with 100 - 120 flies per experiment . The indicated p - value refers to the interaction ( int . ) term of a Cox Proportional Hazards model comparing Abeta42 toxicity ( UAS - A42 vs . ctrl ) and Droj2 expression ( Droj2 + / + vs . Droj2 + / - ) as main factors . The following pairwise comparisons of the indicated groups survival were done by Log Rank test ( * * * * , p < 0 . 0001 ; ns , p > 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05).


( C ) Aversive associative memory performance 2 min after training of aged ( 18 days old ) male Droj2 knockdown flies ( Droj2 + / - ) and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) both expressing human Abeta42 ( UAS - A42 ) of six independent biological replicates . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 6 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test.


( D ) Representative confocal and gSTED deconvolved images of Kenyon cells in 15 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and mitochondrial marker ATP5A - specific antibody ( ATP5A , green ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( E - F ) Solidity ( E ) and circularity ( F ) of ATP5A - stained mitochondria from fly brain gSTED deconvolved images representatively shown in ( D ) from 8 - 10 brains of w1118 wild type ( Droj2 + / + ) and knockdown ( Droj2 + / - ) 15 - day - old male flies . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 - 10 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) Solidity (E) and circularity (F) of ATP5A-stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8-10 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 15-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8-10. *, p < 0.05. Unpaired, two-tailed t-test.


SUMO conjugation assays performed under multiple turnover conditions using wild - type or mutant forms of RPA , SUMO and Siz2 . Only the 80 - minute time points are presented to highlight the formation of Rfa1 - SUMO and Siz2 - SUMO species . The complete time series are presented in Appendix Fig S1 . Bands for Mw marker are fully annotated in Figure 1B .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions using Siz2 ∆ CT and different RPA - DNA complexes . SDS - PAGE gels were stained with Coomassie . Bands for Mw marker are fully annotated in Figure 1B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.


Plot showing the initial rate of SUMO conjugation versus RPA - DNA concentration as obtained from single - turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA - DNA complexes . Representative gels are shown in Appendix Fig S4 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O

Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4.


SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2 ∆ CT lacking ( A ) or including ( B ) its SAP domain . In both cases , reactions were done in technical triplicate and SDS - PAGE gels were stained with SYPRO . Only one representative gel is shown . Bands for Mw marker are fully annotated in Figure 1B .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 . Experiments were performed in technical triplicates ( six times for WT ) . The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2 ∆ CT variants . The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


Electrophoretic mobility shift assays ( EMSA ) to assess binding of RPA or SUMO - RPA to 25nM single - stranded 6 - FAM - labeled 32dT DNA and displacement of SUMO - RPA from 25nM SUMO - RPA bound to single - stranded 6 - FAM - labeled 32dT DNA by RPA and vice versa . All titrations were done in technical triplicates . One representative gel is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electrophoretic mobility shift assays (EMSA) to assess binding of RPA or SUMO-RPA to 25nM single-stranded 6-FAM-labeled 32dT DNA and displacement of SUMO-RPA from 25nM SUMO-RPA bound to single-stranded 6-FAM-labeled 32dT DNA by RPA and vice versa. All titrations were done in technical triplicates. One representative gel is shown.


Electrophoretic mobility shift assays ( EMSA ) performed with 10 nM 6 - FAM - ds20ss32 , a 6 - FAM - labeled 20 - mer dsDNA with a 32 nucleotide 3 ' overhang and its complexes with RPA , RPA with K170 - SUMO modified Rfa1 or RPA reconstituted with ∆ WH - Rfa2 and serially diluted Siz2 . All titrations were done in technical triplicates . One representative gel is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.


A Representative 3 - color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1 / 2 ( presynaptic marker ) , PI ( 3 ) P and Homer ( postsynaptic marker ) . Scale bar , 0 . 5 µm . PI ( 3 ) P - positive structures appear in both pre - and postsynaptic compartments . B Line scan fluorescence intensity profiles of single synapses from the images in A , normalized to maximum value for each channel . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative 3-color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1/2 (presynaptic marker), PI(3)P and Homer (postsynaptic marker). Scale bar, 0.5 µm. PI(3)P-positive structures appear in both pre- and postsynaptic compartments. B Line scan fluorescence intensity profiles of single synapses from the images in A, normalized to maximum value for each channel. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


C Representative 3 - color confocal images of cultured hippocampal neurons fixed and stained for VGlut1 , VGAT and PI ( 3 ) P . Arrows indicate PI ( 3 ) P puncta in VGlut1 + or VGAT + presynaptic terminals . Scale bar , 10 µm . D PI ( 3 ) P intensity in VGlut1 + and VGAT + presynaptic terminals . PI ( 3 ) P is equally abundant in excitatory and inhibitory presynaptic terminals . N = 3 independent experiments ( ≥ 15 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative 3-color confocal images of cultured hippocampal neurons fixed and stained for VGlut1, VGAT and PI(3)P. Arrows indicate PI(3)P puncta in VGlut1+ or VGAT+ presynaptic terminals. Scale bar, 10 µm. D PI(3)P intensity in VGlut1+ and VGAT+ presynaptic terminals. PI(3)P is equally abundant in excitatory and inhibitory presynaptic terminals. N = 3 independent experiments (≥ 15 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


E Relative PI ( 3 ) P levels in the MAP2 + ( Soma ) and Syp + ( Synapse ) areas in DMSO and VPS34IN1 - treated neurons . N = 3 independent experiments ( ≥ 35 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Relative PI(3)P levels in the MAP2+ (Soma) and Syp+ (Synapse) areas in DMSO and VPS34IN1-treated neurons. N = 3 independent experiments (≥ 35 images per condition); Student's t-test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


F Cultured hippocampal neurons were field - stimulated with four trains of 200 APs ( 40 Hz , 5 s ; 90 s gap between each train ) ( Stim ) or left unstimulated ( - ) , fixed and stained for MAP2 , Synaptophysin ( Syp ) , and PI ( 3 ) P . Arrows indicate PI ( 3 ) P puncta in synapses . Scale bar , 10 µm . PI ( 3 ) P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Cultured hippocampal neurons were field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for MAP2, Synaptophysin (Syp), and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. PI(3)P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification.


G Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) from the images in F . N = 5 independent experiments ; ( ≥ 40 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in F. N = 5 independent experiments; (≥ 40 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


H Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) of cultured hippocampal neurons treated with DMSO or Gabazine ( 10 µM ) for 24 h . N = 3 independent experiments ( ≥ 65 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons treated with DMSO or Gabazine (10 µM) for 24 h. N = 3 independent experiments (≥ 65 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


I Cultured hippocampal neurons treated with AP5 ( 20 µM ) , CNQX ( 10 µM ) or TTX ( 0 . 2 µM ) for 24 h , fixed and stained for MAP2 , Syp and PI ( 3 ) P . Arrows indicate PI ( 3 ) P puncta in synapses . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

I Cultured hippocampal neurons treated with AP5 (20 µM), CNQX (10 µM) or TTX (0.2 µM) for 24 h, fixed and stained for MAP2, Syp and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm.


J Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) from the images in I . N ≥ 4 independent experiments ( ≥ 60 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in I. N ≥ 4 independent experiments (≥ 60 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


K Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) of cultured hippocampal neurons were treated with Roscovitine ( 10 µM ) for 1 h and field - stimulated with four trains of 200 APs ( 40 Hz , 5 s ; 90 s gap between each train ) ( Stim ) or left unstimulated ( - ) . N = 3 independent experiments ( ≥ 40 images per condition ) ; Two - way ANOVA ; Fisher ' s Least Significant Difference Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons were treated with Roscovitine (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-). N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Fisher's Least Significant Difference Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


A Basal excitatory synaptic transmission of CA3 - CA1 connections in acute hippocampal slices treated with DMSO ( 0 . 03 % ) or the VPS34 inhibitors , VPS34IN1 ( 3 µM ) or SAR405 ( 20 µM ) . Representative fEPSP traces ( above ) and the relative slope of fEPSPs ( below ) are shown . VPS34 inhibition causes reduced excitatory synaptic transmission down to 46 . 0 ± 4 . 1 % and 55 . 0 ± 4 . 4 % for VPS34IN1 and SAR405 at the end of 60 - min treatment . N = 6 slices per condition from ≥ 4 mice ; Student ' s t - test . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1 % and 55.0 ± 4.4 % for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001


B Increased paired paired - pulse facilitation ( PPF ) at hippocampal CA3 - CA1 synapses after VPS34 inhibition . Representative traces of fEPSPs with a 20 ms interpulse interval is shown above . The ratio of the slope of the second to the first response over a range of interstimulus intervals ( 10 - 500 ms ) is plotted below , indicating an increased facilitation of the second response in VPS34IN1 - treated acute hippocampal slices . PI ( 3 ) P depletion causes a significant facilitation of the second fEPSP , indicative of reduced initial release probability . N = 6 slices per condition from 6 mice ; Two - way ANOVA . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Increased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3)P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001


C Synaptophysin - pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs ( 20 Hz , 1 s ) with 30 s gap between each stimulus . Neurons are acutely treated with DMSO ( 0 . 1 % ) or VPS34 inhibitor1 ( 50 µM ) starting from the onset of 2nd stimulation . The timing of the treatment and the stimulation trains are indicated . D Quantification of data from C . Net difference of Synaptophysin - pHluorin fluorescence 2 s before and 28 s after each stimulation ( 2 s before the next stimulation ) to estimate relative surface retention of SVs . SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1 , leading to progressive accumulation of Synaptophysin - pHluorin signal . N = 4 or 5 independent measurements per condition ; Student ' s t - test . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1 %) or VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation trains are indicated. D Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation (2 s before the next stimulation) to estimate relative surface retention of SVs. SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001


A Electron micrographs of VPS34IN1 - and DMSO - treated synapses following four consecutive trains of 200 AP ( 40 Hz , 5 s ; 90 s gap between each train ) stimulation . The postsynaptic compartment and the postsynaptic density are highlighted in green and purple , respectively . Note the reduced SV numbers and plasma membrane - derived endocytic intermediates in VPS34IN1 - treated synapses . Scale bars , 500 nm ( left ) and 200 nm ( right ) . B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1 . Scale bar , 200 nm . C 3D - reconstruction of a synaptic terminal treated with VPS34IN1 and stimulated as in A . The plasma membrane is colored in turquoise . Synaptic vesicles ( yellow ) , plasma membrane - derived invaginations ( blue ) , endosome - like vacuoles ( green ) and clathrin - coated vesicles ( red ) are highlighted . Scale bar , 500 nm ( top ) and 200 nm ( bottom ) . Data information : Data presented as mean ± SEM n . s . not significant ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Electron micrographs of VPS34IN1- and DMSO-treated synapses following four consecutive trains of 200 AP (40 Hz, 5 s; 90 s gap between each train) stimulation. The postsynaptic compartment and the postsynaptic density are highlighted in green and purple, respectively. Note the reduced SV numbers and plasma membrane-derived endocytic intermediates in VPS34IN1-treated synapses. Scale bars, 500 nm (left) and 200 nm (right). B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1. Scale bar, 200 nm. C 3D-reconstruction of a synaptic terminal treated with VPS34IN1 and stimulated as in A. The plasma membrane is colored in turquoise. Synaptic vesicles (yellow), plasma membrane-derived invaginations (blue), endosome-like vacuoles (green) and clathrin-coated vesicles (red) are highlighted. Scale bar, 500 nm (top) and 200 nm (bottom). Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001


D - H Morphometric quantification of data shown in A . Synapses of VPS34IN1 - treated stimulated neurons display reduced numbers of SVs / bouton area in ( D ) and an accumulation of endocytic membrane invaginations ( E ) , endosome like vacuoles ( ELV ) ( F ) , clathrin - coated pits ( CCP ) ( G ) , and clathrin - coated vesicles ( CCV ) ( H ) . n = 100 ( SV ) and n = 200 ( invagination , ELV , CCP , CCV ) profiles per condition ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-H Morphometric quantification of data shown in A. Synapses of VPS34IN1-treated stimulated neurons display reduced numbers of SVs/ bouton area in (D) and an accumulation of endocytic membrane invaginations (E), endosome like vacuoles (ELV) (F), clathrin-coated pits (CCP) (G), and clathrin-coated vesicles (CCV) (H). n = 100 (SV) and n = 200 (invagination, ELV, CCP, CCV) profiles per condition; Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001


A - D Normalized Synaptophysin - pHluorin responses from neurons treated with VPS34IN1 ( 10 µM for 1h prior to imaging ) and stimulated with four consecutive trains of 50 APs ( 10 Hz , 5 s ) in A or 200 APs ( 40 Hz , 5 s ) in C . PI ( 3 ) P depletion in VPS34IN1 - treated neurons causes the progressive accumulation of Synaptophysin - pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D . Surface retention of Synaptophysin - pHluorin 90 s post - stimulus is plotted for each of the four successive stimulation trains . N ≥ 4 independent experiments ; Two - way ANOVA ; Sidak ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D Normalized Synaptophysin-pHluorin responses from neurons treated with VPS34IN1 (10 µM for 1h prior to imaging) and stimulated with four consecutive trains of 50 APs (10 Hz, 5 s) in A or 200 APs (40 Hz, 5 s) in C. PI(3)P depletion in VPS34IN1-treated neurons causes the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D. Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive stimulation trains. N ≥ 4 independent experiments; Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


E Cultured hippocampal neurons pre - treated with DMSO ( 0 . 1 % ) or VPS34IN1 ( 10 µM , 1 h ) were either field - stimulated with four trains of 200 APs ( 40 Hz , 5 s ; 90 s gap between each train ) ( Stim ) or left unstimulated ( - ) , fixed and stained for surface - stranded Synaptotagmin - 1 , total Synaptotagmin - 1 , and vGlut1 . Scale bar , 10 µm . F Fraction of endogenous Synaptotagmin - 1 stranded on the surface of presynaptic nerve terminals from the images shown in E . N = 3 independent experiments ( ≥ 40 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Cultured hippocampal neurons pre-treated with DMSO (0.1%) or VPS34IN1 (10 µM, 1 h) were either field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for surface-stranded Synaptotagmin-1, total Synaptotagmin-1, and vGlut1. Scale bar, 10 µm. F Fraction of endogenous Synaptotagmin-1 stranded on the surface of presynaptic nerve terminals from the images shown in E. N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


G Surface retention of Synaptophysin - pHluorin 90 s post - stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 ( 20 µM ) . Data are similar to those shown in C and D . N = 3 independent experiments ( ≥ 11 images per condition ) ; Two - way ANOVA ; Sidak ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 (20 µM). Data are similar to those shown in C and D. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


H Exogenous supply of membrane - permeant PI ( 3 ) P acutely rescues defective Synaptophysin - pHluorin endocytosis induced by pharmacological inhibition of VPS34 - mediated PI ( 3 ) P synthesis in hippocampal neurons . Data are similar to those shown in C and D ; where indicated exogenous PI ( 3 ) P - AM ( 20 µM ) was supplied in parallel to VPS34IN1 ( 10 µM ) for 1 h prior to imaging . N = 3 independent experiments ( 15 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Exogenous supply of membrane-permeant PI(3)P acutely rescues defective Synaptophysin-pHluorin endocytosis induced by pharmacological inhibition of VPS34-mediated PI(3)P synthesis in hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3)P-AM (20 µM) was supplied in parallel to VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


I Kinetic block of Synaptophysin - pHluorin endocytosis at hippocampal synapses depleted of VPS34 . Hippocampal neurons depleted of endogenous VPS34 by specific shRNA ( shVPS34 ) or treated with non - targeting control shRNA ( shCtrl ) and stimulated as in C and analyzed as in D . N = 3 independent experiments ( ≥ 10 images per condition ) ; Two - way ANOVA ; Sidak ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ;

O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Kinetic block of Synaptophysin-pHluorin endocytosis at hippocampal synapses depleted of VPS34. Hippocampal neurons depleted of endogenous VPS34 by specific shRNA (shVPS34) or treated with non-targeting control shRNA (shCtrl) and stimulated as in C and analyzed as in D. N =3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001;


A - C The kinetic block of SV endocytosis at excitatory synapses depleted of PI ( 3 ) P depends on prior neuronal activity and Ca2 + . VPS34IN1 - induced defects in SV endocytosis are reverted by parallel treatment with TTX ( 0 . 2 µM ) in A and EGTA ( 2 mM ) in B and AP5 ( 20 µM ) in C , in Synaptophysin - pHluorin - expressing neurons N = 3 independent experiments ( ≥ 10 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C The kinetic block of SV endocytosis at excitatory synapses depleted of PI(3)P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis are reverted by parallel treatment with TTX (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


D Overexpression of dominant - negative Rab5 ( Rab5DN ) , but not constitutively active Rab5 ( Rab5CA ) , rescues the progressive accumulation of Synaptophysin - pHluorin on the surface of VPS34IN1 - treated hippocampal neurons stimulated and analyzed as in A - C . N = 3 independent experiments ( ≥ 10 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


E Levels of various synaptic proteins in DMSO ( 0 . 1 % ) and VPS34IN1 ( 10 µM ) treated cultured cerebellar granule neurons analyzed by western blot . VPS34 inhibition results in p35 processing to hyperactive p25 , a reaction blocked by Calpain inhibitor ALLN ( 100 µM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O

E Levels of various synaptic proteins in DMSO (0.1%) and VPS34IN1 (10 µM) treated cultured cerebellar granule neurons analyzed by western blot. VPS34 inhibition results in p35 processing to hyperactive p25, a reaction blocked by Calpain inhibitor ALLN (100 µM).


F - H Calpain inhibition by ALLN ( 100 µM ) in F or Calpain 2 knockdown via lentiviral shRNA ( shCAPN2 ) in H rescue the progressive accumulation of Synaptophysin - pHluorin on the neuronal surface induced by VPS34IN1 . Calpain 1 knockdown via lentiviral shRNA ( shCAPN1 ) in G had no impact on VPS34IN1 - induced kinetic delay of SV endocytosis . N = 3 independent experiments ( ≥ 11 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H Calpain inhibition by ALLN (100 µM) in F or Calpain 2 knockdown via lentiviral shRNA (shCAPN2) in H rescue the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1. Calpain 1 knockdown via lentiviral shRNA (shCAPN1) in G had no impact on VPS34IN1-induced kinetic delay of SV endocytosis. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


B Cdk5 inhibition by Roscovitine ( Ros , 10 µM ) rescues the progressive accumulation of Synaptophysin - pHluorin on the neuronal surface induced by VPS34IN1 ( 10 µM ) . N = 4 independent experiments ( 20 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparisons Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001


C Calcineurin inhibition by Cyclosporin A ( 10 µM ) phenocopies VPS34IN1 - induced loss of PI ( 3 ) P with respect to the accumulation of Synaptophysin - pHluorin on the neuronal surface after four successive stimulation trains ( 40 Hz , 5 s ) . Data were analyzed as in B . N = 3 independent experiments ( 15 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Calcineurin inhibition by Cyclosporin A (10 µM) phenocopies VPS34IN1-induced loss of PI(3)P with respect to the accumulation of Synaptophysin-pHluorin on the neuronal surface after four successive stimulation trains (40 Hz, 5 s). Data were analyzed as in B. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001


D Basal excitatory synaptic transmission of CA3 - CA1 connections in acute hippocampal slices treated with DMSO ( 0 . 03 % ) , Roscovitine ( Ros , 10 µM ) or VPS34IN1 ( 3 µM ) plus Roscovitine . Representative fEPSP traces ( above ) and the relative slope of fEPSPs ( below ) are shown . Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission , akin to Roscovitine alone , and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%), Roscovitine (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission, akin to Roscovitine alone, and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice; Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001


E Decreased paired paired - pulse facilitation ( PPF ) at hippocampal CA3 - CA1 synapses treated with Roscovitine alone ( Ros , 10 µM ) or VPS34IN1 ( 3 µM ) plus Roscovitine . Representative traces of fEPSPs with a 20 ms interpulse interval is shown above . The ratio of the slope of the second to the first response over a range of interstimulus intervals ( 10 - 500 ms ) is plotted below . The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine - treated acute hippocampal slices , unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice ; Two - way RM ANOVA . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001


A Activity - dependent polyspiking of population spikes ( PS ) during 1 Hz stimulation is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO - treated control slices . Top , representative traces of the 1st and 30th stimulus responses with polyspikes indicated by arrows . N = 12 slices per condition from 12 mice . B Quantification of polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO , VPS34IN1 , or Roscovitine . Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1 . N = 12 slices per condition from 12 mice ; One - way ANOVA . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Activity-dependent polyspiking of population spikes (PS) during 1 Hz stimulation is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO-treated control slices. Top, representative traces of the 1st and 30th stimulus responses with polyspikes indicated by arrows. N = 12 slices per condition from 12 mice. B Quantification of polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO, VPS34IN1, or Roscovitine. Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1. N = 12 slices per condition from 12 mice; One-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.


C The relative changes in the PS and polyspike areas in the presence of picrotoxin ( 50 µM ) and a subsequent treatment with DMSO or VPS34IN1 ( 10 µM ) . VPS34IN1 significantly reduced the PS and polyspike area compared to DMSO . N = 6 slices per condition from 6 mice . D Mean area of PS and polyspikes in picrotoxin - treated slices 40 min post - application of DMSO or VPS34IN1 . N ≥ 6 slices per condition from ≥ 6 mice ; Student ' s t - test . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The relative changes in the PS and polyspike areas in the presence of picrotoxin (50 µM) and a subsequent treatment with DMSO or VPS34IN1 (10 µM). VPS34IN1 significantly reduced the PS and polyspike area compared to DMSO. N = 6 slices per condition from 6 mice. D Mean area of PS and polyspikes in picrotoxin-treated slices 40 min post-application of DMSO or VPS34IN1. N ≥ 6 slices per condition from ≥ 6 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.


A Specific CD8 + T - cell ( OT - I ) proliferation in response to immature BMDCs incubated with fdOVA , fdOVA / sc - αDEC or OVA257 - 264 peptide chemically coupled to anti - DEC - 205 antibody ( NLDC : pOVA ) . Percentage of CFSE - labeled proliferating T cells was evaluated on CD8 + - gated cells after 72 h of DC : T co - culture . The mean ± SD of three independent experiments is reported .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A Specific CD8+ T-cell (OT-I) proliferation in response to immature BMDCs incubated with fdOVA, fdOVA/sc-αDEC or OVA257-264 peptide chemically coupled to anti-DEC-205 antibody (NLDC:pOVA). Percentage of CFSE-labeled proliferating T cells was evaluated on CD8+-gated cells after 72 h of DC:T co-culture. The mean ±SD of three independent experiments is reported.


B Proliferation of adoptively transferred OT - I T cells in C57BL / 6mice ( n = 3 / group ) immunized with vehicle ( PBS ) , NLDC : pOVA , NLDC : pOVA plus CpG , fd , fd / sc - αDEC or LPS - free ovalbuminprotein . Percentage of CFSE - labeled proliferating OT - I T cells was evaluated after three days on Vα2 + CD8 + - gated cells . The mean ± SD of two independent experiments is reported . Comparative analyses were performed using Student ' s t - test for unpaired samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Proliferation of adoptively transferred OT-I T cells in C57BL/6mice (n = 3/group) immunized with vehicle (PBS), NLDC:pOVA, NLDC:pOVA plus CpG, fd, fd/sc-αDEC or LPS-free ovalbuminprotein. Percentage of CFSE-labeled proliferating OT-I T cells was evaluated after three days on Vα2+CD8+-gated cells. The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.


C Percentage of IFN - γ - producing CD8 + splenocytes isolated from mice immunized as in ( B ) . The mean ± SD of two independent experiments is reported . Comparative analyses were performed using Student ' s t - test for unpaired samples .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Percentage of IFN-γ-producing CD8+ splenocytes isolated from mice immunized as in (B). The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.


D - F IL - 6 ( D ) , IL - 18 ( E ) and IFN - α ( F ) release in supernatants of BMDCs obtained from C57BL / 6 mice . Supernatants of BMDCs incubated with NLDC145 antibody , wild - type phage particles ( fdWT ) or scFv αDEC - 205 phage particles ( fdsc - αDEC ) for 20 h were analyzed by ELISA for cytokine production . Unstimulated culture ( medium ) and LPS - treated culture were , respectively , used as negative and positive controls . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6 mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.


D - F IL - 6 ( D ) , IL - 18 ( E ) and IFN - α ( F ) release in supernatants of BMDCs obtained from C57BL / 6mice . Supernatants of BMDCs incubated with NLDC145 antibody , wild - type phage particles ( fdWT ) or scFv αDEC - 205 phage particles ( fdsc - αDEC ) for 20 h were analyzed by ELISA for cytokine production . Unstimulated culture ( medium ) and LPS - treated culture were , respectively , used as negative and positive controls . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.


IL - 6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6 , MYD88 , TLR9 or TLR4 KO mice and incubated for 20 h with wild - type or fdsc - αDEC phage particles . LPS or CpG - ODN were used as controls . IL - 6 release from DCs derived from MyD88 − / − mice was totally abolished and dramatically reduced in DCs derived from Tlr9 − / − mice , but not affected in Tlr4 − / − DCs . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/−mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/−mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.


IFN - α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL / 6 or KO mice and incubated for 20 h with wild - type or fdsc - αDEC phage particles . LPS or CpG - ODN were used as controls . MyD88 − / − and Tlr9 − / − but not Tlr4 − / − BMDCs were unable to produce IFN - α after fdsc - αDEC stimulation . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.


IL - 6 mRNA expression by DC cells . C57BL / 6 , MyD88 − / − and Tlr9 − / − mice were inoculated intraperitoneally with fdWT or fdsc - αDEC bacteriophages or , as a control , with LPS . Mice were sacrificed 2 h later , and purified spleen dendritic cells were analyzed for IL - 6 mRNA levels by quantitative real - time PCR . Bars represent the mean fold increase ± SD . The experiments were performed three times ( n = 2 per group ) . Comparative analyses were performed using Student ' s t - test for unpaired samples .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.


Proliferation of OT - ICD8 + T cells after 72 h of co - culture with 1 × 104DCs isolated from C57BL / 6mice or from MyD88 − / − or Tlr9 − / − transgenic mice previously injected with fd / sc - αDECbacteriophage particles . The panel shows the percentage of divided , CFSE - low OT - ICD8 + T cells . As a control , the proliferation of OT - ICD8 + T cells co - cultured with DCs isolated from non - immunized ( NI ) C57BL / 6mice is reported . The mean ± SD of two independent experiments with n = 3 per group is reported . Comparative analyses were performed using Student ' s t - test for unpaired samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proliferation of OT-ICD8+T cells after 72 h of co-culture with 1 × 104DCs isolated from C57BL/6mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fd/sc-αDECbacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-ICD8+T cells. As a control, the proliferation of OT-ICD8+T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.


IFN - γ production by OT - I CD8 + T cells co - cultured for 12 h in the presence of 3 . 3 × 103 DCs isolated from C57BL / 6 mice or MyD88 − / − and Tlr9 − / − transgenic mice injected as in ( D ) .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).


BMDCs were incubated with TRITC - conjugated fdWT ( A , C ) or fdsc - αDECbacteriophages ( B , D ) for 6 h . Cells were then fixed , permeabilized , immunostained with anti - EEA - 1 ( A , B ) or anti - LAMP - 1 ( C , D ) ( green ) antibody and analyzed by confocalmicroscopy . Representative images are shown . Numbers at the bottom of the right panels indicate the percentage of EEA - 1 ( A , B ) or LAMP - 1 ( C , D ) bacteriophage co - localization ( mean values ± SD , N = 2 , n = 100 ) . Scale bars , 10 mm . White arrows indicate LAMP - 1 - positive structures that also are positive or contain fdsc - αDEC or fdWT bacteriophages .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O

BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDECbacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocalmicroscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.


A - D BMDCs from C57BL / 6mice were stably transduced with YFP - tagged TLR9 . Cells grown on coverslips were then incubated with TRITC - conjugated wild - type ( A , B ) or fdsc - αDEC ( C , D ) bacteriophages for 6 h . Cells were then washed , fixed and analyzed by confocal microscopy . Nuclei were counterstained with Hoechst . ( B , D ) are enlargements of a single cell . Representative images are shown . Numbers at bottom of right panels indicate the percentage of TLR9bacteriophage co - localization ( mean values ± SD , N = 2 , n = 100 ) . Scale bars , 10 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A-D BMDCs from C57BL/6mice were stably transduced with YFP-tagged TLR9. Cells grown on coverslips were then incubated with TRITC-conjugated wild-type (A, B) or fdsc-αDEC (C, D) bacteriophages for 6 h. Cells were then washed, fixed and analyzed by confocal microscopy. Nuclei were counterstained with Hoechst. (B, D) are enlargements of a single cell. Representative images are shown. Numbers at bottom of right panels indicate the percentage of TLR9bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm.


( C - D ) Diverse outcome for tumor cells following interactions with CTLs . OT - I CTLs and OVA + Eµ - myc B cells were labeled with the calcium - sensitive dye Indo - 1 , cultured together in the presence of propidium iodide ( PI ) and subjected to live imaging . Representative time - lapse images ( C ) and corresponding fluorescence quantification ( D ) showing the three distinct outcomes for CTL - tumor cell contacts ( left , calcium response in T cells , and right , calcium and PI signal in targets ) : no detectable damage ( no calcium signal in the target cells ) , reversible damage ( transient calcium elevation and low uptake of PI ) , and irreversible damage ( prolonged calcium elevation and bright PI staining ) . CTLs are outlined by white dashed lines . White arrowheads indicate tumor cells . Red arrowheads indicate tumor cells with internalized PI . Scale bar , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Diverse outcome for tumor cells following interactions with CTLs. OT-I CTLs and OVA+ Eµ-myc B cells were labeled with the calcium-sensitive dye Indo-1, cultured together in the presence of propidium iodide (PI) and subjected to live imaging. Representative time-lapse images (C) and corresponding fluorescence quantification (D) showing the three distinct outcomes for CTL-tumor cell contacts (left, calcium response in T cells, and right, calcium and PI signal in targets): no detectable damage (no calcium signal in the target cells), reversible damage (transient calcium elevation and low uptake of PI), and irreversible damage (prolonged calcium elevation and bright PI staining). CTLs are outlined by white dashed lines. White arrowheads indicate tumor cells. Red arrowheads indicate tumor cells with internalized PI. Scale bar, 5 µm.


( A - B ) Calcium responses were recorded over time in ( A ) unconjugated tumor cells or in ( B ) tumor cells forming a conjugate with a CTL ( during the duration of the contacts ) . Each line corresponds to an individual tumor cell and each square to a time point . Squares are colored in magenta upon calcium elevation or in blue otherwise . Apoptotic events ( detected by caspase 3 activity ) are indicated by an overlaid green dot . Data is representative of three independent experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CTL (during the duration of the contacts). Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.


( A ) Representative time - lapse images showing tumors without CTLs ( upper panels ) or with OT - I CTLs ( lower panels ) . Tumor cells with low and high intracellular calcium appear in blue and red , respectively . CTLs appear in white . Red arrows indicate tumor cells with elevated calcium . Scale bar , 5 µm . Data information : Data were pooled of four independent experiments . ( * p < 0 . 05 , * * * p < 0 . 001 , unpaired t ‑ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative time-lapse images showing tumors without CTLs (upper panels) or with OT-I CTLs (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CTLs appear in white. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm. Data information: Data were pooled of four independent experiments. (*p<0.05, ***p<0.001, unpaired t‑test).


CTLs exhibit extensive differences in lytic activity in vivo . Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct - GFP - expressing OT - I CTLs . Intravital imaging of the bone marrow was performed two days after T cell transfer . ( E ) Representative two - photon time - lapse images showing a representative CTL with high lytic capacity ( killing 4 targets ) and a CTL with no lytic activity for up to 4 hours . Blue arrows mark apoptotic tumor cells . Live and apoptotic tumors appear in grey and blue , respectively . CTLs appear in green . Scale bar , 10 µm . Representative of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CTLs exhibit extensive differences in lytic activity in vivo. Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct-GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. (E) Representative two-photon time-lapse images showing a representative CTL with high lytic capacity (killing 4 targets) and a CTL with no lytic activity for up to 4 hours. Blue arrows mark apoptotic tumor cells. Live and apoptotic tumors appear in grey and blue, respectively. CTLs appear in green. Scale bar, 10 µm. Representative of 3 independent experiments.


( A - B ) Calcium responses were recorded over time in ( A ) unconjugated tumor cells or in ( B ) tumor cells forming a conjugate with a CAR T cell . Each line corresponds to an individual tumor cell and each square to a time point . Squares are colored in magenta upon calcium elevation or in blue otherwise . Apoptotic events ( detected by caspase 3 activity ) are indicated by an overlaid green dot . Data is representative of three independent experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CAR T cell. Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.


( A ) Representative time - lapse images showing tumors without CAR T cells ( upper panels ) or with CAR T Cells ( lower panels ) . Tumor cells with low and high intracellular calcium appear in blue and red , respectively . CAR T cells appear in light green . Red arrows indicate tumor cells with elevated calcium . Scale bar , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative time-lapse images showing tumors without CAR T cells (upper panels) or with CAR T Cells (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CAR T cells appear in light green. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm.


A , Hierarchical clustering analysis of RNA - sequencing data from cells treated with DMSO , 20 Gy IR , 250nM AZD6738 , or AZD6738 + IR ( n = 2 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates).


C , Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR .

C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR.