( B ) Immunoblot of HA - immunoprecipitated lysate of HEK293 co - expressing HA - tagged C9ORF72 / or Flag - tagged SMCR8 / or Flag - tagged WDR41 .

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(B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41.


( C ) Coomassie blue of Nickel - NTA affinity purification of HIS - C9ORF72 , SMCR8 WDR41 co - expressed in baculovirus infected insect .

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(C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells.


( D ) Immunoblot of HA - immunoprecipitated lysate of HEK293 co - expressing HA - tagged C9ORF72 HA - tagged SMCR8 with various Flag - tagged Rab GTPases .

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(D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases.


( E ) Immunoblot against endogenous C9orf72 , Wdr41 , Rab8a , Rab39b , Rab5 Rab7 of control ( IgG alone ) or endogenous Smcr8 immunoprecipitated from adult mouse brain .

(E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain.


( F ) αP32 - radiolabelled GDP release from GST - tagged purified RAB8a as a function of increased of either recombinant purified C9ORF72 alone or in with SMCR8 WDR41 .

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(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.


( G ) Identical GDP release as in ( F ) but using recombinant purified GST - tagged RAB39b instead of RAB8a . Error bars indicate s . e . m . Experiments were repeated 3 times ( n = 3 ) .

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(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).


( A ) Left panel , representative of organotypic cultures of E18 mouse cortical neurons transfected with GFP - RFP - LC3B transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA treated or not with Torin . Right panel , of LC3B .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.


( B ) Upper panel , immunoblot of endogenous LC3B ( Map1lc3b ) , C9orf72 control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA treated or not with Torin . Lower panel , real time RT - qPCR of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA .

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(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.


( C ) Left panel , representative of immunofluorescence labeling of endogenous P62 ( Sqstm1 ) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , of P62 .

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(C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates.


( D ) Left panel , representative of immunofluorescence labeling of transfected constructs ( Green ) endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal transfected with siRNA targeting C9orf72 plasmids expressing control GFP , HA tagged wild type or mutant RAB39b ( CA , Q68L or CN , S22N ) , RAB8a or RAB7 . Right panel , of P62 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot of HA - immunoprecipitated lysate of HEK293 co - expressing HA - tagged C9ORF72 HA - tagged SMCR8 with Flag - tagged TBK1 , NAP1 , TANK or SINTBAD .

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(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.


( B ) Immunoprecipitated HA - tagged C9ORF72 , HA - tagged SMCR8 HA - tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase in presence of γP32 - radiolabelled ATP . were separated by SDS page migration phosphorylation was detected by autoradiography ( upper panel ) , while expression was detected by western blotting ( lower panel ) .

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(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).


( C ) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase of HIS - tagged C9ORF72 : SMCR8 : WDR41 purified from baculovirus infected insect .

(C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.


( D ) Left panel , representative of immunofluorescence labeling of transfected constructs ( Green ) endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal transfected with siRNA targeting 3 ' UTR of Smcr8 mRNA plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 ( TA , S402A T796A ; TD , S402D T796D ; UD , S400D , S492D , S562D T666D ) . Right panel , of P62 .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.


( E ) Left panel , representative of immunofluorescence labeling of transfected constructs ( Green ) endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal transfected with siRNA targeting Tbk1 plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 or RAB39b . Right panel , of P62 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot of endogenous Tdp - 43 , C9orf72 control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA .

(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA.


( B ) Left panel , representative of immunofluorescence labeling of endogenous phosphorylated Ser409 / 410 - Tdp - 43 on cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , of Tdp - 43 .

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(B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates.


( C ) Left panel , representative of immunofluorescence labeling of D169G mutant GFP - tagged TDP - 43 on cultures of E18 mouse cortical neurons transfected with either HA - tagged control or HA - C9ORF72 plasmid . Right panel , of with of TDP - 43 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - , * * * indicates p < 0 . 001 . n = 3 .

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(C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( A ) Left panel , representative of organotypic cultures of E18 mouse cortical neurons co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . Right panel , of ATXN2 . Scale bars , 10 µm . Nuclei were counterstained with DAPI .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI.


( B ) viability ( tetrazolium ) of GT1 - 7 neuronal co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ control siRNA or siRNA targeting C9orf72 mRNA . Error bars indicate s . e . m . Student T - , * * * indicates p < 0 . 001 . n = 3 .

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(B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( C ) Tracing of swimming trajectories of 48 hours post - fertilization zebrafish larvae following touch . ( D - F ) of touch - evoked swimming ( D ) , average velocity ( E ) , maximum velocity attained ( F ) shows significant functional impairment of zebrafish injected with HA - tagged ATXN2 with intermediate of polyQ ( Q30x ) antisense morpholino oligonucleotides ( AMOs ) against C9orf72 compared to control HA - tagged ATXN2 ( Q22x ) or to sole injection of AMO against C9orf72 .

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(C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72.


( G ) Representative of motor neurons visualized with anti - Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone . ( H ) of motor neuron axonal demonstrates a significant decrease of axonal in 48 hours post - fertilization zebrafish larvae injected with both ATXN2 Q30x AMO against C9orf72 compared to controls . Error bars indicate s . e . m . One - way ANOVA , * * indicates p < 0 . 01 . n = 3 .

(G) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.


E F . TLC of cGAMP , which is indicative of cGAS activation , upon incubation with E ) tRNA or F ) total ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . FL - hcGAS was 0 . 265 mg / mL . tRNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , 0 . 85 mg / mL . extracted total concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , 0 . 5 mg / mL . NC indicates negative control that has only FL - hcGAS . dsDNA ISD at a of 0 . 05 mg / mL was used as a positive control . samples were prepared in 20 mM HEPES at pH 7 . 5 150 mM NaCl .

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E and F. TLC analysis of cGAMP, which is indicative of cGAS activation, upon incubation with E) tRNA or F) total RNA (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES at pH 7.5 and 150 mM NaCl.


F . Western blot of OptiPrep gradient fractions with or without DNase RNase treatments .

F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.


G . three on left showing signals of endogenous cGAS in HeLa ( n = 13 , biological replicates , data from one representative independent biological replicate are shown ) . scale bar represents 10 µm . scatterplot on right plots signal intensities of cGAS versus at each pixel in indicated ( yellow circle ) . H . three on left showing signals of mCherry in HEK293T ( n = 9 , biological replicates , data from one representative independent biological replicate are shown ) . scale bar represents 10 µm . scatterplot on right plots signal intensities of mCherry versus at each pixel in indicated ( yellow circle ) . I . Pearson ' s correlation coefficient ( CC ) of signals of cGAS vs . ( n = 13 , biological replicates ) , mCherry vs . ( n = 9 , biological replicates ) . median values were calculated used for evaluation of significance . * * * P < 0 . 001 ( two - Mann - Whitney ) .

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G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).


D . microscopy showing replacement of FTSC - tRNA by Cy5 - ISD at indicated concentrations in preformed granules of hcGAS FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative that is 1 / 16 of raw image is displayed . scale bars represent 20 µm . E . microscopy showing replacement of FTSC - tRNA by TAMRA - 380 bp - dsDNA at indicated concentrations in preformed granules of hcGAS FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative that is 1 / 16 of raw image is displayed . scale bars represent 20 µm . F . of replacement in " D " . For each square , raw image used was evenly divided into four parts . signal ratios of Cy5 - ISD over FTSC - tRNA in four parts were calculated , averaged plotted . G . of replacement in " E " . For each square , raw image used was evenly divided into four parts . signal ratios of TAMRA - 380 bp - dsDNA over FTSC - tRNA in four parts were calculated , averaged plotted .

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D. Fluorescence microscopy images showing the replacement of FTSC-tRNA by Cy5-ISD at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy images showing the replacement of FTSC-tRNA by TAMRA-380 bp-dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in "D". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD over FTSC-tRNA in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in "E". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA over FTSC-tRNA in the four parts were calculated, averaged and plotted.


3 ' - UTR of mUcp1 . Canonical CPEs ( UUUUA1 - 2U in bold ) , reported BRF1 - binding site ( UAUUUAU , gray box ) predicted IMP2 - binding motifs ( AYAYA YA2YA , underlined ) are denoted . Ucp1L Ucp1S carrying long short 3 ' - UTRs , respectively , are resulted from alternative use of two polyadenylation signals ( Hex : AAUAAA , open box ) . ORF , open reading frame . arrowhead marks end of Ucp1S . Northern blots of Ucp1 using 5 µg BAT total denoted radiolabeled probes

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The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes


Schematic comparison of mUcp1L hUcp1 . Poly ( A ) signal ( Hex , open box ) CPE ( black box ) are denoted . RT - PCR results from human , WT ( Ucp1 + / + ) UCP1 - KO ( Ucp1 - / - ) BAT samples

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Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples


Dual luciferase reporter RT - qPCR . reporter plasmids , Fluc - mUcp1S , - mUcp1L or - hUcp1L 3 ' - UTR , Rluc were co - transfected into differentiated HIB1B adipocytes . transfected were used for luciferase activity RT - qPCR . Data are mean ± SD from 3 independent experiments . Translation efficiency was defined as normalized activity ( Fluc / Rluc ) divided by normalized level ( Fluc / Rluc ) . * * * p < 0 . 001 compared with Fluc - mUcp1S , one - way ANOVA

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Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA


Northern blotting of Ucp1 18S rRNA RT - PCR of Ucp1L Ucp1S using total RNAs from denoted BAT samples ( 2 mice per genotype , 2 - 3 mo old ) . short PCR fragments were isolated for sequencing

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Northern blotting of Ucp1 and 18S rRNA and RT-PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples (2 mice per genotype, 2-3 mo old). The short PCR fragments were isolated for sequencing


Western blot using WT Ucp1 ∆ L BAT lysates . level of UCP1 was normalized to GAPDH ( 4 mo old , n = 4 per group ) . * * p < 0 . 01 , Student ' s t

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Western blot analysis using WT and Ucp1∆L BAT lysates. The protein level of UCP1 was normalized to GAPDH (4 mo old, n = 4 per group). **p < 0.01, Student's t test


Dual luciferase reporter . reporter plasmids , Fluc - mUcp1L 3 ' - UTR , Rluc were co - transfected with plasmid expressing EGFP , or myc - tagged CPEB ( immunoblots shown at left ) into differentiated HIB1B adipocytes ( Data from 3 independent experiments ) . * p < 0 . 05 compared with EGFP , one - way ANOVA

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Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1L 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged CPEB (immunoblots shown at the left) into differentiated HIB1B adipocytes (Data from 3 independent experiments). *p < 0.05 compared with EGFP, one-way ANOVA


Western blot using BAT lysates prepared from various CPEB - WT KO ( 4 mo old , n = 4 per group ) . * p < 0 . 05 , Student ' s t

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Western blot analysis using BAT lysates prepared from various CPEB-WT and KO male littermates (4 mo old, n = 4 per group). *p < 0.05, Student's t test


Northern blotting of Ucp1L Ucp1S in BAT from CPEB2 - WT - KO mice at room or after 5 - h cold exposure ( CE ) at 4oC ( 5 mice per genotype , 4 - 7 mo old )

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Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2-WT and -KO male mice at room temperature or after 5-h cold exposure (CE) at 4oC (5 mice per genotype, 4-7 mo old)


Western blot RT - qPCR of mRNA of UCP1 GAPDH in WT KO BAT ( 13 mo old , n = 3 - 4 per group )

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Western blot analysis and RT-qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT (13 mo old, n = 3-4 per group)


Western blot of UCP1 GAPDH in BAT lysates from WT KO mice at 4 12 mo old ( n = 3 per group ) . were normalized to that of GAPDH . RT - qPCR of Ucp1 mRNA level in BAT collected from ( B ) relative to Gapdh mRNA

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Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old (n = 3 per group). Protein levels were normalized to that of GAPDH. RT-qPCR analysis of Ucp1 mRNA level in BAT collected from (B) relative to Gapdh mRNA level


Hematoxylin eosin sections of BAT from WT CPEB2 - KO mice at 4 12 mo old . Scale bars , 1

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Hematoxylin and eosin sections of BAT from WT and CPEB2-KO female mice at 4 and 12 mo old. Scale bars, 1 mm


Lipid droplet number ( i . e . , number of nuclei ) in WT KO BAT ( representative shown in 4D ) were quantified displayed as arbitrary units . Three tissue sections from each BAT 3 mice per group were

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Lipid droplet size and cell number (i.e., number of nuclei) in WT and KO BAT (representative images shown in 4D) were quantified and displayed as arbitrary units. Three tissue sections from each BAT and 3 mice per group were analyzed


representative of WT KO BAT . Three mice per group were

Weight and representative images of WT and KO BAT. Three mice per group were analyzed


CPEB2 - WT - KO mice were transduced with adeno - associated ( AAV ) expressing full - ( AAV _ CP2 ) or - binding domain ( AAV _ RBD ) of CPEB2 . Immunostaining of CPEB2 UCP1 in KO BAT transduced with AAV _ CP2 for 2 , with Hoechst of nuclei . Scale bar , 10 µm . See more in Fig EV4D

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CPEB2-WT and -KO mice were transduced with adeno-associated virus (AAV) expressing full-length (AAV_CP2) or RNA-binding domain (AAV_RBD) of CPEB2. Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV_CP2 for 2 weeks, with Hoechst staining of nuclei. Scale bar, 10 µm. See more images in Fig EV4D


Western blot of UCP1 in BAT lysates from WT KO mice 2 after AAV transduction ( n = 6 mice per group ) . * p < 0 . 05 , Student ' s t

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Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction (n = 6 mice per group). *p < 0.05, Student's t test


BAT of WT KO mice after 2 of AAV transduction measured during 12 - h time ( n = 7 mice per group ) . * p < 0 . 05 , Student ' s t

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BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12-h light time (n = 7 mice per group). *p < 0.05, Student's t test


BAT in WT KO mice , of AAV _ CP2 - transduced WT KO mice ( n = 7 mice per group ) before after i . p . injection of CL316243 ( CL ) . * p < 0 . 05 , repeated - measures two - way ANOVA

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BAT temperature in WT and KO male mice, and of AAV_CP2-transduced WT and KO male mice (n = 7 mice per group) before and after i.p. injection of CL316243 (CL). * p < 0.05, repeated-measures two-way ANOVA


Normalized RPF profile averaged for E . coli transcripts . Profiles generated for grown in absence presence of IPTG ( 1 mM ) . Start stop codons are shaded .

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Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.


Bar chart of measured RBS initiation rates ranked by their strength . Strong RBSs with initiation rates > 1 ribosome / s are highlighted in red . ( D ) Bar chart of measured translation termination efficiencies at stop codons ranked by their strength . Stop codons with translation termination efficiency > 0 . 99 are highlighted in red . ( E ) Distribution of initiation rates for grown in absence presence of IPTG ( 1 mM ) . ( F ) Distribution of translation termination efficiencies for grown in absence presence of IPTG ( 1 mM ) .

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Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).


Comparison of synthesis rate of endogenous E . coli genes measured using Ribo - seq from this study ( in molecules / s units ) from that by Li et al . ( Li et al , 2014 ) ( in molecules / generation units ) . Each point corresponds to a single gene color denotes of transcription initiation rate to translation initiation rate ( giving RNAP / ribosome ) capturing whether transcription ( yellow ) or translation ( blue ) is more dominant .

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Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant.


Transcription ( bottom ) translation ( top ) profiles for yggN , rpoH greA , computed from - seq Ribo - seq data without induction . Positions of genetic parts gene are shown below profiles . ( C ) Promoter strengths in RNAP / s units RBS initiation rates in ribosome / s units .

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Transcription (bottom) and translation (top) profiles for yggN, rpoH and greA, computed from the RNA-seq and Ribo-seq data without induction. Positions of the genetic parts and gene are shown below the profiles. (C) Promoter strengths in RNAP/s units and RBS initiation rates in ribosome/s units.


Transcription ( bottom ) translation ( top ) profiles for lacZ . Profiles are shown for in absence presence of IPTG ( 1 mM ) . Position of genetic parts gene is shown below profiles . RBS is omitted from genetic design due to its . ( E ) Measured promoter strength in RNAP / s units , RBS initiation rate in ribosomes / s units , transcriptional terminator translation termination efficiency for lacZ . Data shown for in absence presence of IPTG ( 1 mM ) .

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Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).


Translation profiles for PK - LacZ construct in cultured in absence ( bottom ) presence ( top ) of IPTG ( 1 mM ) . gene10 , middle , lacZ regions are labelled above profiles . Shaded region denotes PK , dashed lines denote start codon stop codons of gene10 LacZ .

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Translation profiles for the PK-LacZ construct in cells cultured in the absence (bottom) and presence (top) of IPTG (1 mM). The gene10, middle, and lacZ regions are labelled above the profiles. Shaded region denotes the PK, and dashed lines denote the start codon and stop codons of gene10 and LacZ.


Fraction of total RPFs mRNA reads in each reading frame for gene10 , PK or middle , lacZ regions schematically shown below are of PK - LacZ construct . Data shown separately for cultured in absence presence of IPTG ( 1 mM ) .

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Fraction of the total RPFs and mRNA reads in each reading frame for the gene10, PK or middle, and lacZ regions schematically shown below and are of the PK-LacZ construct. Data shown separately for cells cultured in the absence and presence of IPTG (1 mM).


Violin plots of distributions of fractions of total RPFs mRNA reads in each reading frame for E . coli transcripts .

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Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E. coli transcripts.


Change in expression of chromosomal genes in E . coli following induction of PK - lacZ expression ( 1 mM IPTG ) . Each point represents a transcript . Differentially expressed genes ( mRNA : P < 0 . 001 absolute log2 fold - change > 1 . 37 ; translation efficiency : P < 0 . 01 ) are highlighted in color by an alternative point shape ( transcriptional regulation : purple cross ; translational regulation : orange open circle ) .

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Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle).


Translation initiation rates for E . coli RBSs in harboring LacZ PK - LacZ constructs in absence presence of IPTG ( 1 mM ) . Solid shows same initiation rate for both conditions . Dotted lines denote linear regressions for data with no offset .

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Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset.


Fractions of mRNA reads RPFs mapping to each synthetic expression construct ( LacZ PK - LacZ ) E . coli transcripts , which are divided into three major categories : ribosomal , metabolic , other functions . Data shown for cultured in absence presence of IPTG ( 1 mM ) .

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Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).


B HeLa were infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for SEPT7 . scale bar represents 5 μm . Graph represents mean % ± of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 4 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.


C HeLa were infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for p62 . scale bar represents 5 μm . Graph represents mean % ± of Shigella responding to IPTG without ( - ) or with ( + ) p62 from at least 4 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.


D Control ( CTRL ) or isopropanol - treated x - Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . scale bar represents 5 μm .

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D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.


E HeLa were infected for 1 h 30 min , treated with ethanol ( CTRL ) or erythromycin ( EM ) for 2 h prior to fixation , then labelled with antibody for SEPT7 . Graph represents mean % ± of Shigella in SEPT7 cages from 3 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


B HeLa stably expressing SEPT6 - GFP were infected with S . flexneri AfaI for 4 h , harvested , then tested with Co - IP . Empty vector / or uninfected HeLa were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from , extracts were immunoblotted for SEPT6 , GFP , SEPT7 , or p62 . lysates were immunoblotted for GAPDH as control for .

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B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


A HeLa were labelled with MitoTracker Red CMXRos , treated with cytochalasin D for 30 min , then fixed labelled for endogenous SEPT7 for confocal microscopy . scale bar represents 0 . 5 μm . See also Fig EV3A .

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A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.


B HeLa were infected with S . flexneri for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy . scale bar represents 1 μm .

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B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.


C HeLa stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , infected with Shigella - mCherry for 4 h 40 min , processed for CLEM . SEPT6 is shown in green , mitochondria in red , Shigella - mCherry in blue . Septin cages identified by microscopy ( FLM ) were processed for . right - most image is enlarged from boxed region in image , shows bacterium ( Sf ) surrounded by a double of autophagy ( Au ) mitochondrial ( Mt ) . scale bar represents 1 μm . See also Fig EV3B .

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C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.


D HeLa stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , then infected with Shigella - mCherry for 2 h for live confocal microscopy . Time frame shows a septin cage assembling around Shigella . Mitochondria ( circle ) support sites of septin ring assembly around bacterium . Each frame was acquired every 2 min . scale bar represents 1 μm . See also Movie EV1 .

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D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.


E HeLa were treated with control ( CTRL ) , Drp1 , or Mfn1 siRNA for 72 h . Whole lysates were immunoblotted for Drp1 or Mfn1 to show efficiency of depletion . GAPDH was used as loading control . siRNA - treated were infected with S . flexneri for 4 h 40 min , then fixed for microscopy stained for endogenous SEPT7 for quantitative confocal microscopy . Graph represents mean % ± of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment . Student ' s t - , * * = p < 0 . 01 ; * * * = p < 0 . 001 .

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E HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student's t-test, ** = p <0.01; *** = p<0.001.


A HeLa expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos , fixed for confocal microscopy . Inset image highlights location of septins at sites of mitochondrial fission . scale bar represents 5 μm .

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A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.


B HeLa were fixed labelled with antibody against SEPT7 secondary antibody coupled to HRP , processed for . right image is enlarged from boxed region in image , shows a SEPT7 ( S7 ) filament ( labelled in black ) intersecting with mitochondrial ( Mt ) fission site . scale bar represents 100 nm . See also Fig EV4 .

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B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.


C HeLa expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos for live SOFI - super resolution microscopy . Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI - algorithm . Arrows highlight SEPT6 association with location of mitochondrial fission . scale bar represents 1 µm . See also Movie EV3 .

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C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.


D HeLa expressing SEPT6 - GFP were labelled with MitoTracker Red CMXRos , fixed for widefield immunofluorescence microscopy , labelled with antibodies to Drp1 . Arrows highlight one example of Drp1 SEPT6 association with location of mitochondrial fission . scale bar represents 1 μm .

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D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield immunofluorescence microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.


E HeLa were transfected with Mito - BFP ActA - SEPT7 - mCherry , fixed labelled with antibody to Drp1 for widefield immunofluorescence microscopy . scale bar represents 5 μm . Inset highlight Drp1 SEPT7 associated with sites of mitochondrial fission . Pearson ' s correlation coefficient from 3 independent experiments ( mean ± ) shows that mitochondria in ActA - SEPT7 transfected recruit significantly more Drp1 than control ( Mito - BFP transfected alone ) . Student ' s t - , * = p < 0 . 05 .

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E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson's correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student's t-test, * = p<0.05.


A HeLa were treated with control ( CTRL ) , SEPT7 , or Drp1 siRNA for 72 h . Whole lysate of siRNA - treated were immunoblotted for GAPDH , SEPT7 , or Drp1 to show efficiency of siRNA depletion . GAPDH was used as loading control . siRNA - treated were labelled with MitoTracker Red CMXRos , fixed for confocal microscopy . scale bar represents 5 μm .

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A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.


B HeLa were treated with control ( CTRL ) , SEPT2 , SEPT7 , SEPT9 , Drp1 , or SEPT7 + Drp1 siRNA for 72 h , labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Graph represents distribution in ( μm ; whiskers from min to max ) of mitochondria in siRNA - treated from 3 independent experiments ( of at least 100 measurements per biological replicate ) . Student ' s t - , * * * = p < 0 . 001 .

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B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.


C HeLa stably expressing SEPT6 - GFP were harvested , then tested with Co - IP . HeLa expressing GFP were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from , extracts were immunoblotted for Drp1 , SEPT2 , SEPT7 , or GFP . lysates were immunoblotted for GAPDH as control for .

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C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


D HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , whole lysates were immunoblotted for SEPT7 , Drp1 , phosphorylated Drp1 ( P - Drp1 ) , or Mfn1 . GAPDH was used as loading control . Graph represents mean % ± of relative amount of Drp1 , P - Drp1 , Mfn1 , or SEPT7 quantified by densitometry from 4 independent experiments . Student ' s t - , ns = non - significant ; * * = p < 0 . 01 , * * * = p < 0 . 001 .

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D HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student's t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.


E HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed labelled for endogenous P - Drp1 for quantitative confocal microscopy . Representative shown here . scale bar represents 1 μm . recruitment of P - Drp1 to mitochondria was significantly decreased as compared to control as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± , of at least 250 per biological replicate ) . Student ' s t - , * = p < 0 . 05 .

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E HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


F HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed labelled for endogenous Mfn1 for quantitative confocal microscopy . Representative shown here . scale bar represents 1 μm . recruitment of Mfn1 to mitochondria was significantly increased as compared to control as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± , of at least 250 per biological replicate ) . Student ' s t - , * = p < 0 . 05 .

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F HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


A HeLa stably expressing SEPT6 - GFP were treated with Drp1 siRNA for 72 h , transfected with mito - BFP 24 h , then infected with S . flexneri - mCherry for 2 h for live confocal microscopy . Representative image shows interplay between two Shigella - septin cages fused mitochondria . scale bar represents 1 μm . See also Movie EV3 .

A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.


B HeLa were treated with Drp1 siRNA for 72 h , then infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for SEPT7 . Graph represents mean % ± of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


C HeLa were infected with S . flexneri M90T for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Boxplots show of mitochondrial ( μm ; whiskers from min to max ) surrounding Shigella outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments ( of at least 130 measurements per biological replicate ) . Student ' s t - ; * * * = p < 0 . 001 .

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C HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondrial (μm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from 3 independent experiments (analysis of at least 130 measurements per biological replicate). Student's t-test; *** = p<0.001.


D HeLa were either kept uninfected as a control or infected with S . flexneri M90T or S . flexneri ΔIcsA for 4 h 40 min . Samples were labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Boxplots show of mitochondria ( μm ; whiskers from min to max ) in uninfected ( CTRL ) , surrounding S . flexneri M90T ( + M90T ) or S . flexneri ΔIcsA ( + ΔIcsA ) from 3 independent experiments ( of at least 250 measurements per biological replicate ) . Student ' s t - ; * * * = p < 0 . 001 .

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D HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ΔIcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (μm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ΔIcsA (+ΔIcsA) from 3 independent experiments (analysis of at least 250 measurements per biological replicate). Student's t-test; *** = p<0.001.


CSF PGRN is increased in mutation carriers ( MC ) compared to non - carriers ( NC ) .

CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC).


CSF PGRN is not associated with in either NC or MC .

CSF PGRN is not associated with age in either NC or MC.


CSF PGRN is increased in males compare to females.


CSF PGRN do not differ among MC participants carrying a PSEN1 , PSEN2 or APP mutation .

CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.


CSF PGRN as a function of EYO in mutation carriers ( MC , red ) non - carriers ( NC , blue ) . solid lines indicate regression for each of groups 95 % confidence interval ( CI ) calculated by a linear model adjusting by gender . interaction term of mutation status EYO is significant ( P = 0 . 041 ) , also when including PGRN outliers participants with EYO > + 20 ( P = 0 . 030 ) . data points are not displayed to prevent disclosure of mutation status .

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CSF PGRN as a function of EYO in mutation carriers (MC, red) and non-carriers (NC, blue). The solid lines indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender. The interaction term of mutation status and EYO is significant (P = 0.041), also when including PGRN outliers and participants with EYO > +20 (P = 0.030). Individual data points are not displayed to prevent disclosure of mutation status.


graph depicts standardised differences of CSF PGRN between MCs NCs as a function of EYO , in context of other biomarker cognitive changes . curves were generated by linear model that best fit each marker ( see statistics section Appendix Table S2 ) . CSF PGRN is significantly increased in MC compared to NC 10 years before expected symptom onset ( shadowed ) after brain amyloidosis brain injury ( as measured by CSF T - tau ) have started , shortly before CSF sTREM2 starts to increase . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; CSF , cerebrospinal fluid ; MC , mutation carriers ; MMSE , Mini - Mental State Examination ; NC , non - carriers ; T - tau , total tau .

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The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO, in the context of other biomarker and cognitive changes. The curves were generated by the linear model that best fit each marker (see statistics section and Appendix Table S2). CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset (shadowed area) after brain amyloidosis and brain injury (as measured by CSF T-tau) have started, and shortly before CSF sTREM2 starts to increase. Abbreviations: Aβ1-42: amyloid-β 42; CSF, cerebrospinal fluid; MC, mutation carriers; MMSE, Mini-Mental State Examination; NC, non-carriers; T-tau, total tau.


Scatter plot representing of CSF PGRN in healthy controls ( depicted in blue ) different stages of Alzheimer ' s continuum ( depicted in red ) . graphs were performed excluding CSF PGRN outliers ( 1 ' healthy control ' , 1 ' Preclinical AD A + / TN - ' , 4 ' AD CDR = 0 . 5 ' 1 ' AD CDR = 1 ' ) . Including them yielded a similar result CSF PGRN was still significantly higher in ' AD CDR = 1 ' group compared to ' healthy controls ' ( P = 0 . 001 ) ' Preclinical AD A + / TN - ' ( P = 0 . 0001 ) groups . Abbreviations : A : amyloid - β biomarker status ; AD : Alzheimer ' s disease ; CDR : clinical dementia rating ; CSF , cerebrospinal fluid ; N : neurodegeneration biomarker status ; T : tau pathology biomarker status .

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Scatter plot representing the levels of CSF PGRN in healthy controls (depicted in blue) and the different stages of the Alzheimer's continuum (depicted in red). The analysis and graphs were performed excluding CSF PGRN outliers (1 'healthy control', 1 'Preclinical AD A+/TN-', 4 'AD CDR = 0.5' and 1 'AD CDR = 1'). Including them yielded a similar result and CSF PGRN was still significantly higher in the 'AD CDR = 1' group compared to the 'healthy controls' (P = 0.001) and 'Preclinical AD A+/TN-' (P = 0.0001) groups. Abbreviations: A: amyloid-β biomarker status; AD: Alzheimer's disease; CDR: clinical dementia rating; CSF, cerebrospinal fluid; N: neurodegeneration biomarker status; T: tau pathology biomarker status.


Scatter plots representing association of CSF PGRN with different cognitive . Only subjects of Alzheimer ' s continuum group ( n = 474 ) were included . In studied , higher of CSF PGRN were associated with worse cognitive performance ( namely , lower scores in ADNI - Mem , ADNI - EF MMSE higher scores in ADAS - Cog11 , ADAS - Cog13 CDR - SB ) . graphs are excluding PGRN outliers ; including them rendered similar results ( Appendix Table S6 ) . Abbreviations : ADAS - Cog , Alzheimer ' s disease Assessment Scale - cognitive subscale ; ADNI - Mem : ADNI memory composite score ; ADNI - EF : ADNI executive function composite score ; CDR - SB : clinical dementia rating Sum of Boxes ; MMSE , Mini - Mental State Examination .

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Scatter plots representing the association of CSF PGRN with different cognitive tests. Only the subjects of the Alzheimer's continuum group (n = 474) were included. In all tests studied, higher levels of CSF PGRN were associated with worse cognitive performance (namely, lower scores in ADNI-Mem, ADNI-EF and MMSE and higher scores in ADAS-Cog11, ADAS-Cog13 and CDR-SB). The analysis and the graphs are excluding PGRN outliers; including them rendered similar results (Appendix Table S6). Abbreviations: ADAS-Cog, Alzheimer's disease Assessment Scale - cognitive subscale; ADNI-Mem: ADNI memory composite score; ADNI-EF: ADNI executive function composite score; CDR-SB: clinical dementia rating Sum of Boxes; MMSE, Mini-Mental State Examination.


Scatter plots representing association of CSF PGRN with temporo - parietal FDG - PET uptake within subjects of Alzheimer ' s continuum group ( n = 474 ) . Abbreviations : FDG - PET : Fludeoxyglucose positron emission

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Scatter plots representing the association of CSF PGRN with temporo-parietal FDG-PET uptake within the subjects of the Alzheimer's continuum group (n = 474). Abbreviations: FDG-PET: Fludeoxyglucose positron emission tomography


Scatter plots representing association of CSF PGRN with total hippocampal volume ( B ) within subjects of Alzheimer ' s continuum group ( n = 474 ) .

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Scatter plots representing the association of CSF PGRN with total hippocampal volume (B) within the subjects of the Alzheimer's continuum group (n = 474).


Scatter plots representing associations of CSF PGRN with CSF sTREM2 each of AD CSF biomarkers ( T - tau , P - tau181P Aβ1 − 42 ) in non - carriers ( NC , blue ; A , C , E G ) in mutation carriers ( MC , red ; B , D , F H ) . Each point depicts value of CSF PGRN corresponding biomarker of a subject solid lines indicate regression 95 % confidence interval ( CI ) for each of groups . standardized regression coefficients ( β ) P - values are shown were computed using a linear model adjusting for , gender APOE ε4 . sample contained some outliers ( defined as 3 SDs below or above group mean ) of CSF markers of AD . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 .

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Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.


Scatter plots representing associations of CSF PGRN with CSF sTREM2 each of AD CSF biomarkers ( T - tau , P - tau181P Aβ1 − 42 ) in healthy controls ( blue ; A , D , G J ) , Alzheimer ' s continuum ( red ; B , E , H K ) ' suspected non - Alzheimer ' s pathophysiology ( SNAP ) ' groups ( green ; C , F , I L ) . Each point depicts value of CSF PGRN corresponding biomarker of a subject solid lines indicate regression 95 % confidence interval ( CI ) for each of groups . standardized regression coefficients ( β ) P - values are shown were computed using a linear model adjusting for , gender APOE ε4 . sample contained some outliers ( defined as 3 SDs below or above group mean ) of CSF markers of AD , including these outliers yielded similar results ( Appendix Table S8 ) . Aβ1 - 42 values used for associations are those on an extrapolation curve since upper technical limit is 1700pg / ml . We also tested associations with Aβ1 - 42 values truncated at upper technical limit result was similar . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 ; SNAP : suspected non - Alzheimer ' s pathophysiology .

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Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in healthy controls (blue; A, D, G and J), Alzheimer's continuum (red; B, E, H and K) and 'suspected non-Alzheimer's pathophysiology (SNAP)' groups (green; C, F, I and L). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD, the analysis including these outliers yielded similar results (Appendix Table S8). The Aβ1-42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg/ml. We also tested the associations with Aβ1-42 values truncated at the upper technical limit and the result was similar. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181; SNAP: suspected non-Alzheimer's pathophysiology.


( A ) Representative Western Blot of endogenous p150Glued co - immunoprecipitated with EB1 in shCTL or shSTIM1 ECs ( left ) its by normalized densitometry ( right ) . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty mouse IgG . Results are average ± SD of three independent assays . shCTL value of each biological replicate was normalized on itself so shSTIM1 experimental value . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL shSTIM1 p ≤ 0 , 05 * .

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(A) Representative Western Blot analysis of the endogenous p150Glued co-immunoprecipitated with EB1 in shCTL or shSTIM1 ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty mouse IgG. Results are the average ± SD of three independent assays. shCTL value of each biological replicate was normalized on itself and so shSTIM1 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL and shSTIM1 p≤ 0,05 *.


( B ) Representative of three Western Blot of endogenous p150Glued , - intermediate chain ( LIC ) of dynein 1 EB1 immunoprecipitated with STIM1 in wild - type ECs . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty rabbit IgG .

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(B) Representative of three Western Blot analysis of endogenous p150Glued, light-intermediate chain (LIC) of cytoplasmic dynein 1 and EB1 immunoprecipitated with STIM1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG.


( D ) Representative Western Blot of increasing amount of GST - STIM1 ( domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in absence or presence of FLAG - EB1 C - terminal . Colors label as in ( c ) . Negative control was performed using equal amount of empty GST , together with Cap - Gly - CC1 - p150Glued .

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(D) Representative Western Blot analysis of increasing amount of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in absence or presence of FLAG-EB1 C-terminal. Colors label the protein as in (c). Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.


( E ) Representative Western Blot of mCherry - p150Glued WT co - immunoprecipitated with GFP - STIM1 WT , NN , ΔCC1 - 3 / WT ( ΔCC / WT ) or ΔCC1 - 3 / NN ( ΔCC / NN ) in co - transfected HEK 293T . Negative control ( CTL ) was performed incubating lysate from HEK 293T co - transfected with an empty GFP vector together with mCherry - P150Glued WT with pre - cleared A or G - Sepharose rabbit GFP antibody . Below , its by normalized densitometry . Results are average ± SD of three independent assays . value of P150Glued co - immunoprecipitated with GFP - STIM1 WT from each biological replicate was normalized on itself so those immunoprecipitated with GFP - STIM1 NN , ΔCC / WT or ΔCC / NN . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT NN p > 0 . 05 not significant ( ns ) , for STIM1 WT ΔCC / WT p > 0 . 05 not significant ( ns ) for STIM1 WT ΔCC / NN p ≤ 0 , 05 * .

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(E) Representative Western Blot analysis of mCherry-p150Glued WT co-immunoprecipitated with GFP-STIM1 WT, NN, ΔCC1-3/WT (ΔCC/WT) or ΔCC1-3/NN (ΔCC/NN) in co-transfected HEK 293T cells. Negative control (CTL) was performed incubating cell lysate from HEK 293T co-transfected with an empty GFP vector together with mCherry-P150Glued WT with pre-cleared protein A or G-Sepharose and the rabbit GFP antibody. Below, its quantification by normalized densitometry. Results are the average ± SD of three independent assays. The value of P150Glued co-immunoprecipitated with GFP-STIM1 WT from each biological replicate was normalized on itself and so those immunoprecipitated with GFP-STIM1 NN, ΔCC/WT or ΔCC/NN. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p> 0.05 not significant (ns), for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns) and for STIM1 WT and ΔCC/NN p≤ 0,05 *.


( F ) Representative Western Blot of GST - STIM1 ( domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in presence of FLAG - EB1 C - terminal WT or ΔY . Negative control was performed using equal amount of empty GST , together with Cap - Gly - CC1 - p150Glued .

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(F) Representative Western Blot analysis of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in presence of FLAG-EB1 C-terminal WT or ΔY. Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.


( G ) Confocal microscopy of wild - type ECs , transiently transfected with GFP - STIM1 WT or , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) together with mCherry - P150Glued . Scale bar = 10 μm . Right insets are shown to highlight MT - bound p150Glued + ( arrows ) , colocalized with STIM1 . Scale bar = 5 μm . ( H ) Average number of mCherry - P150Glued + in same as in ( g ) . are average ± of three independent experiments for a total of 150 ( 30 ) . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT NN p ≤ 0 , 001 * * * for STIM1 WT ΔCC / WT p > 0 . 05 not significant ( ns ) . ( I ) Co - localization of mCherry - P150Glued with GFP - STIM1 WT , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) , transiently co - transfected in ECs , as in ( g ) . Results are average ± of three independent experiments for a total of 150 ( 30 ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT NN p ≤ 0 , 001 * * * for STIM1 WT ΔCC / WT p > 0 . 05 not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy analysis of wild-type ECs, transiently transfected with GFP-STIM1 WT or, NN or ΔCC1-3/WT (ΔCC/WT) together with mCherry-P150Glued. Scale bar = 10 μm. Right insets are shown to highlight MT-bound p150Glued+ punctae (arrows), colocalized with STIM1. Scale bar = 5 μm. (H) Average number of the mCherry-P150Glued+ punctae in the same cells as in (g). Counts are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells). Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p≤ 0,001 *** and for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns). (I) Co-localization analysis of mCherry-P150Glued with GFP-STIM1 WT, NN or ΔCC1-3/WT (ΔCC/WT), transiently co-transfected in ECs, as in (g). Results are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p≤ 0,001 *** and for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns).


( G ) Confocal microscopy of wild - type ECs , transiently transfected with GFP - STIM1 WT or , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) together with mCherry - P150Glued . Scale bar = 10 μm . Right insets are shown to highlight MT - bound p150Glued + ( arrows ) , colocalized with STIM1 . 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

(G) Confocal microscopy analysis of wild-type ECs, transiently transfected with GFP-STIM1 WT or, NN or ΔCC1-3/WT (ΔCC/WT) together with mCherry-P150Glued. Scale bar = 10 μm. Right insets are shown to highlight MT-bound p150Glued+ punctae (arrows), colocalized with STIM1. Scale bar = 5 μm.


( A ) Confocal microscopy of ECs silenced with a control siRNA ( siCTL ) or one targeting STIM1 ( siSTIM1 ) stained for endogenous LAMP - 1 ( in green ) to visualize LEs DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . 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

(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous LAMP-1 (in green) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( B ) Distribution of , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from three independent experiments for a total of 249 endosomes ( 15 siCTL ) 441 endosomes ( 13 siSTIM1 ) . In right inset , cumulative distribution used for Kolmogorov - Smirnov , p ≤ 0 , 001 * * * , is shown . p - value refers to rejection of null hypothesis in favor of alternative hypothesis that siCTL LE distribution has longer tail than siSTIM1 LE one .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of size, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 249 endosomes (15 siCTL cells) and 441 endosomes (13 siSTIM1 cells). In the right inset, the cumulative distribution used for the Kolmogorov-Smirnov test, p≤ 0,001 ***, is shown. The p-value refers to the rejection of the null hypothesis in favor of the alternative hypothesis that siCTL LE distribution has longer tail than siSTIM1 LE one.


( C ) Average number , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are average ± of three independent experiments for a total of 249 late endosomes in 15 siCTL ( 16 ± 2 endosomes per ) 441 late endosomes in 13 siSTIM1 ( 34 ± 3 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , 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

(C) Average number, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of three independent experiments for a total of 249 late endosomes in 15 siCTL cells (16 ± 2 endosomes per cell) and 441 late endosomes in 13 siSTIM1 cells (34 ± 3 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( D ) Distribution of to nucleus , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from three independent experiments for a total of 249 late endosomes in 15 siCTL ( 16 ± 2 endosomes per ) 441 late endosomes in 13 siSTIM1 ( 34 ± 3 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

(D) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 249 late endosomes in 15 siCTL cells (16 ± 2 endosomes per cell) and 441 late endosomes in 13 siSTIM1 cells (34 ± 3 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( E ) Confocal microscopy of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , rescued for GFP expression alone ( shCTL / GFP shSTIM1 / GFP ) or for GFP - STIM1 WT ( shSTIM1 / GFP - STIM1 WT ) , GFP - STIM1 NN ( shSTIM1 / GFP - STIM1 NN ) or GFP - STIM1 AA ( shSTIM1 / GFP - STIM1 AA ) stained for endogenous LAMP - 1 ( in red ) to visualize LEs DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . 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

(E) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), rescued for GFP expression alone (shCTL/GFP and shSTIM1/GFP) or for GFP- STIM1 WT (shSTIM1/GFP-STIM1 WT), GFP- STIM1 NN (shSTIM1/GFP-STIM1 NN) or GFP- STIM1 AA (shSTIM1/GFP-STIM1 AA) and stained for endogenous LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( F ) Upper panel , Distribution of to nucleus , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from of two independent experiments for a total of 436 late endosomes in 13 shCTL / GFP ( 34 ± 6 endosomes per ) , 1452 late endosomes in 12 shSTIM1 / GFP ( 121 ± 13 endosomes per ) 498 late endosomes in 10 shSTIM1 / GFP - STIM1 WT ( 50 ± 5 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * ( orange ) for shSTIM1 / GFP shSTIM1 / GFP - STIM1 WT p ≤ 0 , 001 * * * ( green ) . Lower panel , Distribution of to nucleus , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from of two independent experiments for a total of 498 late endosomes in 10 shSTIM1 / GFP - STIM1 WT ( 50 ± 5 endosomes per ) , 1057 late endosomes in 11 shSTIM1 / GFP - STIM1 NN ( 96 ± 12 endosomes per ) 346 late endosomes in 10 shSTIM1 / GFP - STIM1 AA ( 35 ± 2 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for shSTIM1 / GFP - STIM1 WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 05 * ( orange ) for shSTIM1 / GFP - STIM1 NN shSTIM1 / GFP - STIM1 AA p ≤ 0 , 05 * ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper panel, Distribution of distance to nucleus, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell) and 498 late endosomes in 10 shSTIM1/GFP-STIM1 WT cells (50 ± 5 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 *** (orange) and for shSTIM1/GFP and shSTIM1/GFP-STIM1 WT p≤ 0,001 *** (green). Lower panel, Distribution of distance to nucleus, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from of two independent experiments for a total of 498 late endosomes in 10 shSTIM1/GFP-STIM1 WT cells (50 ± 5 endosomes per cell), 1057 late endosomes in 11 shSTIM1/GFP-STIM1 NN cells (96 ± 12 endosomes per cell) and 346 late endosomes in 10 shSTIM1/GFP-STIM1 AA cells (35 ± 2 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for shSTIM1/GFP-STIM1 WT and shSTIM1/GFP-STIM1 NN p≤ 0,05 * (orange) and for shSTIM1/GFP-STIM1 NN and shSTIM1/GFP-STIM1 AA p≤ 0,05 * (green).


( G ) Average , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are average ± of two independent experiments for a total of 436 late endosomes in 13 shCTL / GFP ( 34 ± 6 endosomes per ) , 1452 late endosomes in 12 shSTIM1 / GFP ( 121 ± 13 endosomes per ) , 615 late endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 51 ± 5 endosomes per ) , 1278 late endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 106 ± 15 endosomes per ) 506 late endosomes in 12 shSTIM1 / GFP - STIM1 AA ( 42 ± 7 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * , for shCTL / GFP shSTIM1 / GFP - STIM1 WT p > 0 . 05 not significant ( ns ) , for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * , for shSTIM1 / GFP shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 AA p > 0 . 05 not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average size, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell), 615 late endosomes in 12 shSTIM1/GFP-STIM1 WT cells (51 ± 5 endosomes per cell), 1278 late endosomes in 12 shSTIM1/GFP-STIM1 NN cells (106 ± 15 endosomes per cell) and 506 late endosomes in 12 shSTIM1/GFP-STIM1 AA cells (42 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 ***, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p> 0.05 not significant (ns), for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,001 ***, for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,001 *** and for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 AA p> 0.05 not significant (ns).


( H ) Average number , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are average ± of two independent experiments for a total of 436 late endosomes in 13 shCTL / GFP ( 34 ± 6 endosomes per ) , 1452 late endosomes in 12 shSTIM1 / GFP ( 121 ± 13 endosomes per ) , 615 late endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 51 ± 5 endosomes per ) , 1278 late endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 106 ± 15 endosomes per ) 506 late endosomes in 12 shSTIM1 / GFP - STIM1 AA ( 42 ± 7 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * , for shCTL / GFP shSTIM1 / GFP - STIM1 WT p ≤ 0 , 001 * * * , for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 05 * , for shSTIM1 / GFP shSTIM1 / GFP - STIM1 NN p ≤ 0 , 01 * * for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 AA p > 0 . 05 not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average number, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell), 615 late endosomes in 12 shSTIM1/GFP-STIM1 WT cells (51 ± 5 endosomes per cell), 1278 late endosomes in 12 shSTIM1/GFP-STIM1 NN cells (106 ± 15 endosomes per cell) and 506 late endosomes in 12 shSTIM1/GFP-STIM1 AA cells (42 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 ***, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p≤ 0,001 ***, for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,05 *, for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,01 ** and for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 AA p> 0.05 not significant (ns).