A 10 min time - lapse imaging of DIV12 wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO + cald - GFP ) or caldendrin knock - out treated with jasplakinolide ( cald KO + JPK ) mouse primary hippocampal neurons , additionally expressing a cell fill ( maxGFP ) and an ER - marker ( ER - tDimer ) . Yellow arrows indicate protrusions with a stably anchored ER ( > 10 min ) . Blue arrows indicate protrusions that show transient presence of ER . Scale bar = 5 µm .

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

A 10 min time-lapse imaging of DIV12 wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO + cald-GFP) or caldendrin knock-out treated with jasplakinolide (cald KO + JPK) mouse primary hippocampal neurons, additionally expressing a cell fill (maxGFP) and an ER-marker (ER-tDimer). Yellow arrows indicate protrusions with a stably anchored ER (>10 min). Blue arrows indicate protrusions that show transient presence of ER. Scale bar = 5 µm.


A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin . The groups compared are wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO cald ) , and caldendrin knock - out treated with jasplakinolide ( cald KO JPK ) . Scale bar = 5 µm .

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A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO cald), and caldendrin knock-out treated with jasplakinolide (cald KO JPK). Scale bar = 5 µm.


B - C Quantification of spine density , synaptopodin clusters and synaptopodin - positive spines as shown in A ( mean ± SEM . Kruskal - Wallis test with Dunn ' s multiple comparisons post - test . * * * p < 0 . 0001 . cald wt n = 34 cells , 2 experiments ; cald KO n = 33 cells , 3 experiments ; cald KO cald n = 10 cells , 1 experiment ; cald KO JPK n = 14 cells , 1 experiment ) . ( B ) The total number of synaptopodin clusters is unaffected by the loss of caldendrin . ( C ) The number of synaptopodin - positive spines is reduced in cald KO compared to the WT neurons . This phenotype could be rescued by over - expression of caldendrin ( cald KO + cald ) , but not by treatment with JPK ( cald KO + JPK ) .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_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 B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O

B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald KO n = 33 cells, 3 experiments; cald KO cald n = 10 cells, 1 experiment; cald KO JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK).


Media from soluble TREM2 - Fc transfected HEK293T cells incubated with Aβ42 fibrils . Top left panel shows original media samples . Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet . The bottom two panels represent the same samples as the top , but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils . Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet . The bottom two panels represent the same samples as the top , but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1 . All experiments were replicated 3 independent

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Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM2 - Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Left panels show original media samples . Right panels show the purified soluble TREM2 - Fc ( bottom ) and the presence of APOE3 with the purified soluble TREM2 - Fc ( top ) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1 . All experiments were replicated 3 independent

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Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM - Fc family member transfected HEK293T cells incubated with Aβ42 fibrils . Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet . Bottom panel shows original media and Aβ42 pellet probed with

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Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10


Media from soluble TREM - Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3 . Bottom panel shows original media and purified soluble TREM - Fc family members probed with anti -

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Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5


Schematic representation of Aβ42 oligomers ( right panel ) and APOE4 ( left panel ) binding with soluble TREM2 - Fc at various concentrations . Curves were fit by setting buffer control ( no ligand ) to y =

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Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0


BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2 - Fc pre incubated with Aβ42 oligomers . Schematic representations of two sequential associations of ligand on soluble TREM2 - Fc are represented by I and II . Soluble TREM2 - Fc loaded on a sensor is first incubated with APOE3 ( association I ) followed by a second incubation with Aβ42 oligomers ( association II ) or the reverse - incubated with Aβ42 oligomers ( association I ) followed by a second incubation with APOE3 ( association II ) . All experiments were replicated 3 independent times

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

BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2-Fc pre incubated with Aβ42 oligomers. Schematic representations of two sequential associations of ligand on soluble TREM2-Fc are represented by I and II. Soluble TREM2-Fc loaded on a sensor is first incubated with APOE3 (association I) followed by a second incubation with Aβ42 oligomers (association II) or the reverse- incubated with Aβ42 oligomers (association I) followed by a second incubation with APOE3 (association II). All experiments were replicated 3 independent times


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2 - Fc shows a dose dependent signal . Results were expressed as the OD450 averaged ± standard error ( n = 3 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

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

ELISA plates were coated with purified soluble TREM2-Fc, and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2-Fc shows a dose dependent signal. Results were expressed as the OD450 averaged ± standard error (n =3; * p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM - Fc family members . Results were expressed as the OD450 averaged ± standard error ( n = 4 , * p < 0 . 05 , * * * * p < 0 . 0001 , ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

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ELISA plates were coated with purified soluble TREM2-Fc, and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM-Fc family members. Results were expressed as the OD450 averaged ± standard error (n = 4, * p < 0.05, **** p<0.0001, ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


2B4 NFAT - GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours . GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage ± GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with mock , * * * * p < 0 . 0001 compared with mock , # p < 0 . 05 compared with TREM2 , # # # # p < 0 . 0001 compared with TREM2 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S3 for p value )

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2B4 NFAT-GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours. GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage ± GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with mock, **** p< 0.0001 compared with mock, # p < 0.05 compared with TREM2, #### p < 0.0001 compared with TREM2, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S3 for p value)


B - 2B4 NFAT - GFP reporter cells transduced with TREM2 or variants ( R47H , R62H and T96K ) were incubated with 10 µM Aβ42 oligomers for 12 hours . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with TREM2 , * * p < 0 . 05 compared with TREM2 , * * * * p < 0 . 0001 compared with TREM2 , # # p < 0 . 01 compared with TREM2 T96K , # # # # p < 0 . 0001 compared with TREM2 T96K , ANOVA , Dunnett ' s multiple comparisons test , see Appendix table S3 for p value

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

B-2B4 NFAT-GFP reporter cells transduced with TREM2 or variants (R47H, R62H and T96K) were incubated with 10 µM Aβ42 oligomers for 12 hours. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with TREM2, ** p < 0.05 compared with TREM2, **** p< 0.0001 compared with TREM2, ## p < 0.01 compared with TREM2 T96K, #### p < 0.0001 compared with TREM2 T96K, ANOVA, Dunnett's multiple comparisons test, see Appendix table S3 for p value


HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA . Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant . Results were averaged ± standard error ( n = 9 , # p < 0 . 05 compared with TREM2 , # # p < 0 . 01 compared with TREM2 , * * * p < 0 . 001 compared with mock , * * * * p < 0 . 0001 compared with mock , ANOVA , Tukey ' s Multiple Comparison Test , see Appendix table S3 for p value )

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HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA. Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant. Results were averaged ± standard error (n = 9, # p < 0.05 compared with TREM2, ## p< 0.01 compared with TREM2, *** p< 0.001 compared with mock, **** p <0.0001 compared with mock, ANOVA, Tukey's Multiple Comparison Test, see Appendix table S3 for p value)


HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours . Conditioned media and cell lysate were analyzed by Western blot for TREM2 ( RIPA lysate ) and soluble TREM2 ( media ) detection . Experiment was replicated 3 independent

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HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours. Conditioned media and cell lysate were analyzed by Western blot for TREM2 (RIPA lysate) and soluble TREM2 (media) detection. Experiment was replicated 3 independent times


HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification . Bottom panel in D shows the presence of TREM2 in the purified product . Endogenous APP and β - tubulin were used as controls for the purification ( middle and upper panels ) . Experiment was replicated 3 independent

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HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification. Bottom panel in D shows the presence of TREM2 in the purified product. Endogenous APP and β-tubulin were used as controls for the purification (middle and upper panels). Experiment was replicated 3 independent time


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen , scale bar 100

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(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm


( B Quantification of GLUT1 ( hypoxia marker Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e .

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(B Quantification of GLUT1 (hypoxia marker Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m


( C ) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

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(C) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


, D ) Quantification o CD31 ( endothelial cell marker ) . Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e . m . (

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,D) Quantification o CD31 (endothelial cell marker). Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m. (C


( E , F ) Expression levels of DEGs relevant to response to hypoxia ( left ) and blood vessel morphogenesis ( right ) . The values were normalized by tubulin family

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(E,F) Expression levels of DEGs relevant to response to hypoxia (left) and blood vessel morphogenesis (right). The values were normalized by tubulin family genes


( G ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen , scale bar 100

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(G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm


( H ) Quantification of HIF - 1A in PDAC tissues Control ( n = 5 ) , and 5mg ( n = 4 ) . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum

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(H) Quantification of HIF-1A in PDAC tissues Control (n=5), and 5mg (n=4). In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value


( I ) qPCR levels of HIF - 1A in PSCs , normalized to RPLP0 and relative to

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(I) qPCR levels of HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Western blot bands for protein expression in PSCs ( p - Tmod is post - translational modification ) . The plot shows the quantification of the sum of band intensities corresponding to isoform 1 , isoform 2 , isoform 3 , and post - transcriptionally modified HIF - 1A ( n = 8 control and n = 8 tam )

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(J) Western blot bands for protein expression in PSCs (p-Tmod is post-translational modification). The plot shows the quantification of the sum of band intensities corresponding to isoform 1, isoform 2, isoform 3, and post-transcriptionally modified HIF-1A (n=8 control and n=8 tam)


( A ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 and relative to

(A) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot levels of LOX - L2 in PSCs ( n = 3 experimental replicates , * * P < 0 . 01 )

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(B) Western blot levels of LOX-L2 in PSCs (n=3 experimental replicates, **P<0.01)


( C ) Expression of LOX family genes obtained from RNA - seq data in control and tamoxifen treated PSCs ( n = 3 experimental replicates ) . Expression value was normalized by tubulin family

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(C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes


( D ) qPCR levels of LOX family in PSCs , normalized to RPLP0 and relative to

(D) qPCR levels of LOX family in PSCs, normalized to RPLP0 and relative to control


( E ) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in

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(E) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates


( F ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 50 μm . ( G ) Quantification of LOX - L2 for images in F . n ≥ 4 mice per condition , and n > 10 sections per

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(F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal


( H ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to 1

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(H) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to 1 kPa


( I ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to

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(I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Quantification of average forces applied by PSCs on pillars . BBI = blebbistatin . In the box and whisker plot , the central box represents values from the lower and upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum . * * * P < 0 . 001 Three experimental

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(J) Quantification of average forces applied by PSCs on pillars. BBI = blebbistatin. In the box and whisker plot, the central box represents values from the lower and upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum. ***P<0.001 Three experimental repeats


( K ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 ( 60S acidic ribosomal protein P0 ) and relative to

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(K) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 (60S acidic ribosomal protein P0) and relative to control


( A ) Images of matrigel collagen gels previously remodelled by PSCs , second harmonic generation signal for fibrillar collagen ( green ) and F - actin ( red ) , scale bar 100 μm . ( B ) Fiber thickness colour - code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness , scale bar 100 μm . ( C ) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm . Insets show FFTs of fibrillar collagen - I images , representing alignment with respect to the elliptical distribution of the FFT central maxima . Circular behaviour ( values approaching 1 ) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction . Scale bar 20 μm ( D - F ) Quantification of fiber thickness , length , and alignment for images in A - C . ( G ) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B , H . n ≥ 6 matrices per condition . In the scattered plots each point represents a section . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum value . Histogram bars represent mean ± s . e . m . * * * P < 0 . 001 ( t - test for D , E , F and ; Anova and Tukey " s post hoc test for G ) . Three experimental replicates for all

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(A) Images of matrigel collagen gels previously remodelled by PSCs, second harmonic generation signal for fibrillar collagen (green) and F-actin (red), scale bar 100 μm. (B) Fiber thickness colour-code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness, scale bar 100 μm. (C) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm. Insets show FFTs of fibrillar collagen-I images, representing alignment with respect to the elliptical distribution of the FFT central maxima. Circular behaviour (values approaching 1) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction. Scale bar 20 μm (D-F) Quantification of fiber thickness, length, and alignment for images in A-C. (G) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B,H. n≥6 matrices per condition. In the scattered plots each point represents a section. In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value. Histogram bars represent mean±s.e.m. ***P<0.001 (t-test for D, E, F and ; Anova and Tukey"s post hoc test for G). Three experimental replicates for all p


( A ) qPCR levels of collagen in PSCs , normalized to RPLP0 and relative to

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(A) qPCR levels of collagen in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot analysis of collagen normalised to total protein and relative to

(B) Western blot analysis of collagen normalised to total protein and relative to control


( C ) Quantification of time - lapse collagen synthesis and deposition by

(C) Quantification of time-lapse collagen synthesis and deposition by PSCs


( D ) Representative immunofluorescent images used for the quantification in C , scale bar 20 μm , collagen - I was assessed with a specific primary antibody

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(D) Representative immunofluorescent images used for the quantification in C, scale bar 20 μm, collagen-I was assessed with a specific primary antibody staining


( E ) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

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(E) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


( F ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to

(F) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to contro


( G ) MMP - 2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography ; above signal intensity of the representative bands used for the quantification presented in the plot

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(G) MMP-2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography; above signal intensity of the representative bands used for the quantification presented in the plot below


quantification of MMP - 2 levels in PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 100 μm ( n = 4 animals per condition and n ≥ 5 sections per animal )

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quantification of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 100 μm (n=4 animals per condition and n≥ 5 sections per animal)


Immunohistochemistry image of MMP - 2 levels in PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of

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Immunohistochemistry image of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxife


( J ) Relative values of protein levels for MMP - 2 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

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(J) Relative values of protein levels for MMP-2 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


( K ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to 1

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(K) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to 1 kPa


( L ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to control . Histogram bars represent mean ± s . e . m .

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(L) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to control. Histogram bars represent mean±s.e.m.


( A ) qPCR levels of fibronectin ( FN ) , fibronectin extracellular domain A ( FN - EDA ) , and fibronectin extracellular domain B ( FN - EDB ) in PSCs , normalised to RPLP0 and relative to

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(A) qPCR levels of fibronectin (FN), fibronectin extracellular domain A (FN-EDA), and fibronectin extracellular domain B (FN-EDB) in PSCs, normalised to RPLP0 and relative to control


( B ) Western blot analysis of FN normalised to total protein and relative to

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(B) Western blot analysis of FN normalised to total protein and relative to control


( C ) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by

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(C) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs


( D ) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by

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(D) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs


( E ) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control , and 2mg , and 5mg of tamoxifen . ( n = 4 animals per condition , and n ≥ 5 sections per animal ) . ( F ) FN fiber thickness colour - code map in E represented through the BoneJ

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(E) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control, and 2mg, and 5mg of tamoxifen. (n=4 animals per condition, and n ≥ 5 sections per animal). (F) FN fiber thickness colour-code map in E represented through the BoneJ plugin


( G ) Relative values of protein levels for FN in PDAC tumors assessed by proteomic

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(G) Relative values of protein levels for FN in PDAC tumors assessed by proteomic analysis


( H - J ) Quantification of fibronectin immunohistochemistry staining , fiber thickness and alignment scored of images presented in e . In all cases , histogram bars represent mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test for A , B , C ; Anova and Tukey " s post hoc test for H , I , J ) . Scale bar is

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(H-J) Quantification of fibronectin immunohistochemistry staining, fiber thickness and alignment scored of images presented in e. In all cases, histogram bars represent mean ± s.e.m.; *P<0.05,**P<0.01,***P<0.001 (t-test for A, B, C; Anova and Tukey"s post hoc test for H, I, J). Scale bar is 1


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control , and 2mg of tamoxifen , scale bar is 100 μm . Upper panels : Ki67 staining is used as a surrogate of proliferation . White arrows indicate Ki67 positive nuclei in epithelial cells . Lower panels : Cc3 staining shows the cells undergoing caspase - mediated apoptosis . Tamoxifen panels show higher levels of yellow staining , which indicates higher percentage of apoptotic epithelial cells . ( B ) Quantification of staining in panel A ( n = 4 animals per condition , and n ≥ 5 sections per animal , two experimental repetitions ) . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t -

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(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control, and 2mg of tamoxifen, scale bar is 100 μm. Upper panels: Ki67 staining is used as a surrogate of proliferation. White arrows indicate Ki67 positive nuclei in epithelial cells. Lower panels: Cc3 staining shows the cells undergoing caspase-mediated apoptosis. Tamoxifen panels show higher levels of yellow staining, which indicates higher percentage of apoptotic epithelial cells. (B) Quantification of staining in panel A (n=4 animals per condition, and n≥ 5 sections per animal, two experimental repetitions). Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test


( A , E , F ) Immunofluorescence images of control and tamoxifen treated suit - 2 cells , scale bar is 20 μm . Panel A represents HIF - 1A staining in hypoxia and non - hypoxia conditions , panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase - mediated apoptosis , respectively . Panel E : Red - F - actin , green - Ki67 , blue - nuclei . Tamoxifen negatively regulates HIF - 1A in hypoxia and non - hypoxia conditions . ( B , C , D ) Quantification of immunofluorescence staining in panels A , E , F . For quantification , eight fields of view ( n > 50 cells ) per condition . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t - test . All panels include data collected during 3 independent

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(A, E, F) Immunofluorescence images of control and tamoxifen treated suit-2 cells, scale bar is 20 μm. Panel A represents HIF-1A staining in hypoxia and non-hypoxia conditions, panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase-mediated apoptosis, respectively. Panel E: Red - F-actin, green - Ki67, blue - nuclei. Tamoxifen negatively regulates HIF-1A in hypoxia and non-hypoxia conditions. (B,C,D) Quantification of immunofluorescence staining in panels A, E, F. For quantification, eight fields of view (n>50 cells) per condition. Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test. All panels include data collected during 3 independent experiments


A Montage of class averages and 3D volumes of humanTFG ( amino acids 1 ‐ 193 ; EMDataBank accession code EMD ‐ 6076 ) . Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference ‐ free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left . Scale bars , 50 Å .

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A Montage of class averages and 3D volumes of humanTFG (amino acids 1‐193; EMDataBank accession code EMD‐6076). Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference‐free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left. Scale bars, 50 Å.


B Montage of class averages and 3D volumes of C . elegans TFG ( amino acids 1 ‐ 195 ; EMDataBank accession code EMD ‐ 6075 ) , generated as described in ( A ) . Scale bars , 50 Å .

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B Montage of class averages and 3D volumes of C. elegans TFG (amino acids 1‐195; EMDataBank accession code EMD‐6075), generated as described in (A). Scale bars, 50 Å.


C Superposition of 3D volumes of full ‐ length human TFG and truncated TFG ( amino acids 1 ‐ 193 ) . The truncated form is depicted in yellow , while the full ‐ length form is depicted as gray mesh . The top view ( top ) exhibits limited differences in the structures , while the side view ( bottom ) shows extra density in the full ‐ length TFG isoform . Scale bar , 25 Å .

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C Superposition of 3D volumes of full‐length human TFG and truncated TFG (amino acids 1‐193). The truncated form is depicted in yellow, while the full‐length form is depicted as gray mesh. The top view (top) exhibits limited differences in the structures, while the side view (bottom) shows extra density in the full‐length TFG isoform. Scale bar, 25 Å.


A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations . Error bars represent mean ± SEM ; n = 10 replicates .

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A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations. Error bars represent mean ± SEM; n = 10 replicates.


B Confocal microscopy images of recombinant , BODIPY ‐ labeled TFG in the presence of varying potassium acetate concentrations . Scale bar , 2 μm .

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B Confocal microscopy images of recombinant, BODIPY‐labeled TFG in the presence of varying potassium acetate concentrations. Scale bar, 2 μm.


C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain ‐ EM . Large arrowheads highlight TFG polymers ( only found in the presence of elevated potassium acetate ) . Small arrowheads highlight individual 11 nm cup ‐ like TFG octamers . Scale bar , 30 nm .

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C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain‐EM. Large arrowheads highlight TFG polymers (only found in the presence of elevated potassium acetate). Small arrowheads highlight individual 11 nm cup‐like TFG octamers. Scale bar, 30 nm.


D Circular dichroism spectroscopy was used to characterize the carboxyl ‐ terminus of C . elegans TFG ( amino acids 195 ‐ 486 ) . Samples were analyzed at different concentrations , and the data were normalized relative to one another . CD spectra were collected at 25 ° C in 25 mM sodium phosphate ( pH 7 . 2 ) using a 1 mm path length quartz cell . The spectra are characteristic of an intrinsically disordered protein .

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D Circular dichroism spectroscopy was used to characterize the carboxyl‐terminus of C. elegans TFG (amino acids 195‐486). Samples were analyzed at different concentrations, and the data were normalized relative to one another. CD spectra were collected at 25°C in 25 mM sodium phosphate (pH 7.2) using a 1 mm path length quartz cell. The spectra are characteristic of an intrinsically disordered protein.


A The distributions of endogenous TFG and ERGIC ‐ 53 were examined in hTERT ‐ RPE ‐ 1 cells stably expressing low levels of mApple ‐ Sec16B using SR ‐ SIM ( more than 15 cells analyzed on 3 separate occasions ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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A The distributions of endogenous TFG and ERGIC‐53 were examined in hTERT‐RPE‐1 cells stably expressing low levels of mApple‐Sec16B using SR‐SIM (more than 15 cells analyzed on 3 separate occasions). Scale bar, 5 μm. Inset scale bar, 1 μm.


B Cells stably expressing low levels of GFP ‐ Sec16B and mRuby ‐ Sec23A ( Sec23A ) were fixed and imaged using SR ‐ SIM ( n = 12 ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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B Cells stably expressing low levels of GFP‐Sec16B and mRuby‐Sec23A (Sec23A) were fixed and imaged using SR‐SIM (n = 12). Scale bar, 5 μm. Inset scale bar, 1 μm.


C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby ‐ Sec23A ( Sec23A ) using SR ‐ SIM ( n = 10 ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby‐Sec23A (Sec23A) using SR‐SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm.


D The distributions of endogenous TFG and Sec31A were examined using g ‐ STED ( n = 8 ) . Scale bar , 1 μm .

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D The distributions of endogenous TFG and Sec31A were examined using g‐STED (n = 8). Scale bar, 1 μm.


E The distributions of endogenous TFG and Sec31A were examined using immunogold EM . Large arrowheads highlight 15 nm gold particles bound to α ‐ TFG antibodies . The small arrowhead highlights 5 nm gold particles bound to α ‐ Sec31A antibodies . At least 15 different cells were examined , and representative images are shown . Scale bar , 100 nm .

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E The distributions of endogenous TFG and Sec31A were examined using immunogold EM. Large arrowheads highlight 15 nm gold particles bound to α‐TFG antibodies. The small arrowhead highlights 5 nm gold particles bound to α‐Sec31A antibodies. At least 15 different cells were examined, and representative images are shown. Scale bar, 100 nm.


A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes ( including full ‐ length , untagged TFG , a truncation mutant encoding amino acids 1 ‐ 300 of untagged TFG , and a truncation mutant encoding amino acids 1 ‐ 350 of untagged TFG ) and stained using α ‐ TFG and α ‐ Sec31A antibodies . Scale bar , 5 μm . Inset scale bar , 1 μm . Images shown are representative of at least 15 individual cells analyzed for each condition .

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A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes (including full‐length, untagged TFG, a truncation mutant encoding amino acids 1‐300 of untagged TFG, and a truncation mutant encoding amino acids 1‐350 of untagged TFG) and stained using α‐TFG and α‐Sec31A antibodies. Scale bar, 5 μm. Inset scale bar, 1 μm. Images shown are representative of at least 15 individual cells analyzed for each condition.


B A recombinant polyhistidine ‐ and SUMO ‐ tagged TFG fragment ( Sumo ‐ TFGc , amino acids 194 ‐ 400 ; total molecular mass of 42 kDa ) was expressed and purified from Escherichia coli extracts using nickel affinity resin . Coomassie ‐ stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column ( top ) or a 10 - 30 % glycerol gradient ( bottom ) are shown . To determine the native molecular weight of the protein , the following equation was used : M = 6πηNas / ( 1 − υρ ) , where M is the native molecular weight , η is the viscosity of the medium , N is Avogadro ' s number , a is the Stokes radius , s is the sedimentation value , υ is the partial specific volume , and ρ is the density of the medium ( Siegel Monty , ) . These data suggest that this fragment of TFG is capable of forming dimers in solution .

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B A recombinant polyhistidine‐ and SUMO‐tagged TFG fragment (Sumo‐TFGc, amino acids 194‐400; total molecular mass of 42 kDa) was expressed and purified from Escherichia coli extracts using nickel affinity resin. Coomassie‐stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column (top) or a 10-30% glycerol gradient (bottom) are shown. To determine the native molecular weight of the protein, the following equation was used: M = 6πηNas/(1 − υρ), where M is the native molecular weight, η is the viscosity of the medium, N is Avogadro's number, a is the Stokes radius, s is the sedimentation value, υ is the partial specific volume, and ρ is the density of the medium (Siegel Monty, ). These data suggest that this fragment of TFG is capable of forming dimers in solution.


C , D Control cells ( C ) or cells depleted of endogenous TFG ( D ) were transfected with a construct expressing TFG with a carboxyl ‐ terminal GFP tag and stained using α ‐ Sec31A antibodies . Scale bar , 5 μm . Images shown are representative of at least 20 individual cells analyzed for each condition .

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C, D Control cells (C) or cells depleted of endogenous TFG (D) were transfected with a construct expressing TFG with a carboxyl‐terminal GFP tag and stained using α‐Sec31A antibodies. Scale bar, 5 μm. Images shown are representative of at least 20 individual cells analyzed for each condition.


A HumanRPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . Cells were fixed and stained using α ‐ Sec16A and α ‐ Sec31A antibodies and imaged using confocal microscopy . Images shown are projections of 3D data sets ( 4 μm in z ) . Both individual and merged images with Sec16A in green and Sec31A in red are shown ( at least 15 cells for each condition ) . Scale bar , 5 μm . Higher magnification views of the indicated regions ( boxed ) are also shown in the lower or upper right portion of each image . Inset scale bar , 1 μm . B Quantification of the number of Sec31A ‐ labeled structures in a 4 μm2 region . For both conditions ( control or TFG siRNA ‐ treated ) , at least 50 distinct regions away from the peri ‐ nuclear Golgi were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ test .

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A HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐Sec31A antibodies and imaged using confocal microscopy. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and Sec31A in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Inset scale bar, 1 μm.B Quantification of the number of Sec31A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.


C Quantification of the number of Sec16A ‐ labeled structures in a 4 μm2 region . For both conditions ( control or TFG siRNA ‐ treated ) , at least 50 distinct regions away from the peri ‐ nuclear Golgi were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . No statistically significant difference was observed , following analysis using a paired t ‐ test .

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C Quantification of the number of Sec16A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. No statistically significant difference was observed, following analysis using a paired t‐test.


D Cells stably expressing low levels of GFP ‐ ERGIC ‐ 53 and mRuby ‐ Sec23A were fixed and imaged using SR ‐ SIM ( top ) , or depleted of endogenous TFG prior to fixation and SR ‐ SIM imaging ( bottom ) . Images shown are representative of at least 15 individual cells analyzed for each condition . Scale bar , 5 μm . Inset scale bar , 1 μm .

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D Cells stably expressing low levels of GFP‐ERGIC‐53 and mRuby‐Sec23A were fixed and imaged using SR‐SIM (top), or depleted of endogenous TFG prior to fixation and SR‐SIM imaging (bottom). Images shown are representative of at least 15 individual cells analyzed for each condition. Scale bar, 5 μm. Inset scale bar, 1 μm.


E HumanRPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . Cells were fixed and stained using α ‐ Sec16A and α ‐ ERGIC ‐ 53 antibodies and imaged using SR ‐ SIM . Images shown are projections of 3D data sets ( 4 μm in z ) . Both individual and merged images with Sec16A in green and ERGIC ‐ 53 in red are shown ( at least 15 cells for each condition ) . Scale bar , 5 μm . Higher magnification views of the indicated regions ( boxed ) are also shown in the lower or upper right portion of each image . Arrowheads highlight Sec16A ‐ labeled structures that do not exhibit juxtaposed ERGIC ‐ 53 staining . Inset scale bar , 1 μm . F Based on structured illumination microscopy , quantification of the number of Sec16A ‐ labeled structures juxtaposed ( within 500 nm ) to ERGIC ‐ 53 ‐ labeled structures is shown . For both conditions ( control or TFG siRNA ‐ treated ) , at least 1 , 000 unique Sec16A ‐ labeled structures were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ test .

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E HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐ERGIC‐53 antibodies and imaged using SR‐SIM. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and ERGIC‐53 in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Arrowheads highlight Sec16A‐labeled structures that do not exhibit juxtaposed ERGIC‐53 staining. Inset scale bar, 1 μm.F Based on structured illumination microscopy, quantification of the number of Sec16A‐labeled structures juxtaposed (within 500 nm) to ERGIC‐53‐labeled structures is shown. For both conditions (control or TFG siRNA‐treated), at least 1,000 unique Sec16A‐labeled structures were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.


A HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple ( ManII ‐ mApple ) were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed and stained using TFG antibodies and imaged using confocal microscopy . Alternatively , cells were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with TFG in green and ManII in red are shown . Scale bar , 5 μm . Images shown are representative of at least 10 individual cells analyzed for each condition . B Human RPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy . Alternatively , cells were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with TFG in green and GM130 in red are shown . Scale bar , 5 μm . Images shown are representative of at least 10 individual cells analyzed for each condition .

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A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple (ManII‐mApple) were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and ManII in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.B Human RPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and GM130 in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.


C Higher magnification views of the indicated regions in ( B , boxed ) are shown ( GM130 distribution only ) . Scale bar , 2 μm .

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C Higher magnification views of the indicated regions in (B, boxed) are shown (GM130 distribution only). Scale bar, 2 μm.


A HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . Cells were subsequently treated with brefeldin A ( BFA ) for 1 h , followed by a wash into fresh media and further incubation for 2 h in the absence of BFA , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with Sec31A in green and ManII in red are shown ( representative of at least 15 cells analyzed ) . Scale bar , 5 μm .

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A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.


B HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . Cells were subsequently treated with BFA for 1 h , followed by a wash into fresh media and further incubation for 1 h in the absence of BFA , prior to fixation and staining using α ‐ Sec16A and α ‐ Sec31A antibodies . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with Sec31A ( green ) , ManII ( red ) , and Sec16A ( blue ) are shown ( representative of at least 15 cells analyzed ) . Scale bar , 5 μm . C Higher magnification views of the indicated regions in ( B , boxed ) are shown . Arrows highlight COPII ‐ positive transport carriers that contain the cargo ManII , which are not juxtaposed to Sec16A ‐ labeled sites on the ER . Additionally , arrowheads point out distinct foci in which COPII continues to associate with Sec16A ‐ labeled sites , indicating that COPII vesicle formation continues in the absence of TFG . Scale bar , 1 μm .

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B HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.C Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.


Formalin - fixed paraffin - embedded ( FFPE ) sections from control or Tgif1KO mice were immunostained with anti - pSmad2 antibody and revealed by immunofluorescence ( IF ) and DAPI . Representative pictures at 40x are shown ( n = 30 ) . Scale bars , 100 μM .

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Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM.


FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin ( H & E ) or immunostained with antibodies to CK19 or Amylase and revealed by IHC . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Kaplan - Meyer survival analysis of control , Tgif1KO , KC and KTC mice . A regular mosaic two - colors line was used to discriminate between control and Tgif1KO mice .

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Kaplan-Meyer survival analysis of control, Tgif1KO, KC and KTC mice. A regular mosaic two-colors line was used to discriminate between control and Tgif1KO mice.


Pictures of whole pancreas and spleen tissues from control , Tgif1KO , KC or KTC mice ( left ) . Weight of pancreas was measured and presented as dot plot ( n = 30 ) .

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Pictures of whole pancreas and spleen tissues from control, Tgif1KO, KC or KTC mice (left). Weight of pancreas was measured and presented as dot plot (n=30).


FFPE pancreatic sections from control , Tgif1KO , KC or KTC were stained with H & E or subjected to IHC analysis using antibodies to CK19 or α - SMA . Yellow arrows indicate PanINs in KC mice . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control, Tgif1KO, KC or KTC were stained with H&E or subjected to IHC analysis using antibodies to CK19 or α-SMA. Yellow arrows indicate PanINs in KC mice. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Representative pictures of dissected abdominal region of KC and KTC mice . Yellow arrows indicate metastatic nodules in liver . Yellow dotted lines show the pancreas .

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Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas.


FFPE sections of pancreas , liver , lungs or lymph - nodes from control , KC or KTC mice were stained with H & E or immunostained with anti - Vimentin antibody and revealed by IF . Representative pictures at 4x ( Lymph node , H & E ) , 10x ( Liver , H & E ) and 20x ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) are shown ( n = 18 to 30 ) . Scale bars , 1 , 000 μM ( Lymph node , H & E ) , 400 μM ( Liver , H & E ) and 200 μM ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) .

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FFPE sections of pancreas, liver, lungs or lymph-nodes from control, KC or KTC mice were stained with H&E or immunostained with anti-Vimentin antibody and revealed by IF. Representative pictures at 4x (Lymph node, H&E), 10x (Liver, H&E) and 20x (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin) are shown (n=18 to 30). Scale bars, 1,000 μM (Lymph node, H&E), 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin).


Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence . Following imaging live animals , pancreas , brain , liver and lung were dissected and analyzed again by IVIS bioluminescence ( n = 6 , 5 out of 6 mice developed metastasis to liver and lung ) .

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Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence. Following imaging live animals, pancreas, brain, liver and lung were dissected and analyzed again by IVIS bioluminescence (n= 6, 5 out of 6 mice developed metastasis to liver and lung).


Total cell lysates from pancreas of control , KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2 , Smad2 or JunB ( n = 6 ) .

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Total cell lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2, Smad2 or JunB (n=6).


Expression of JunB in pancreas from control , KC or KTC mice ( same as in D ) was analyzed by qRT - PCR ( n = 6 ) . Data are expressed as mean ± SEM . * * p < 0 . 01 ; based on a two - tailed Student ' s test .

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Expression of JunB in pancreas from control, KC or KTC mice (same as in D) was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; based on a two-tailed Student's test.


Protein extracts from MIAPaCa - 2 cells ( left ) and control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 ( A - C ) .

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Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C).


Protein extracts from control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading , total cell lysates were analyzed by immunoblotting using antibodies to Twist1 , TGIF1 or β - Actin .

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Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.


Protein extracts from MIAPaCa - 2 cells ( left ) and control were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C , cell lysates were treated with DNase I before coimmunopreciptation .

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Protein extracts from MIAPaCa-2 cells (left) and control were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C, cell lysates were treated with DNase I before coimmunopreciptation.


Panc - 1 cells were treated with TGF - β for 30 min , and protein extracts were immunoprecipitated with anti - TGIF1 antibody and analyzed by immunoblotting using anti - Twist1 antibody . To control for activation of TGF - β signaling , total cell lysates were analyzed by immunoblotting using anti - pSmad2 antibody .

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Panc-1 cells were treated with TGF-β for 30 min, and protein extracts were immunoprecipitated with anti-TGIF1 antibody and analyzed by immunoblotting using anti-Twist1 antibody. To control for activation of TGF-β signaling, total cell lysates were analyzed by immunoblotting using anti-pSmad2 antibody.


PL45 stably expressing Dox - inducible control or sh - RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) . In left panel , the intensity of the bands was assessed by densitometry , and the result was presented as a ratio of Twist1 / TGIF1 .

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PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1.


PL45 stably expressing Dox - inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel .

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PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel .

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Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


PL45 stably expressing both Dox - inducible TGIF1 and Tam - inducible Twist1 ( Twist1ER ) were treated with Tam and / or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) .

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PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).


MIAPaCa - 2 cells were transfected with wild - type or mutated ( Twist1 binding site ) Twist1 luciferase reporters ( TGTLuc ) together with increasing amounts of Twist1 expression vector in the presence of empty vector ( EV ) or TGIF1 expression vector . Luciferase activity was measured 48 hr following transfection and normalized on the basis of co - transfected Renilla luciferase ( n = 6 ) .

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MIAPaCa-2 cells were transfected with wild-type or mutated (Twist1 binding site) Twist1 luciferase reporters (TGTLuc) together with increasing amounts of Twist1 expression vector in the presence of empty vector (EV) or TGIF1 expression vector. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).


Total lysates from pancreas of control , KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E - Cadherin , Vimentin , p16Ink4A and β - Actin ( n = 6 ) .

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Total lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E-Cadherin, Vimentin, p16Ink4A and β-Actin (n=6).


Expression of Cdh1 , Vimentin , and p16Ink4A in pancreas from control , KC or KTC mice ( same as in C ) was analyzed by qRT - PCR ( n = 6 ) .

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Expression of Cdh1, Vimentin, and p16Ink4A in pancreas from control, KC or KTC mice (same as in C) was analyzed by qRT-PCR (n=6).


FFPE pancreatic sections from control , KC or KTC mice were stained with H & E or immunostained with antibodies to E - Cadherin , Vimentin or p16Ink4a and revealed by IF or IHC . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Kaplan - Meyer survival analysis of control , KTC and KTWC mice .

Kaplan-Meyer survival analysis of control, KTC and KTWC mice.


FFPE sections of pancreas or liver from control , KTC or KTWC mice were stained with H & E or immunostained with anti - CK19 antibody and revealed by IHC . Representative pictures at 10x ( Liver , H & E ) or 20x ( Pancreas , H & E ; Pancreas , IHC - CK19 ) are shown ( n = 30 ) . Scale bars , 400 μM ( Liver , H & E ) and 200 μM ( Pancreas , H & E ; Pancreas , IHC - CK19 ) .

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FFPE sections of pancreas or liver from control, KTC or KTWC mice were stained with H&E or immunostained with anti-CK19 antibody and revealed by IHC. Representative pictures at 10x (Liver, H&E) or 20x (Pancreas, H&E; Pancreas, IHC-CK19) are shown (n=30). Scale bars, 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Pancreas, IHC-CK19).


Expression of Cdh1 , Vimentin and p16Ink4A in pancreas from control , KTC or KTWC mice was analyzed by qRT - PCR ( n = 6 ) .

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Expression of Cdh1, Vimentin and p16Ink4A in pancreas from control, KTC or KTWC mice was analyzed by qRT-PCR (n=6).


Specificity of anti - phospho - TGIF1 ( pTGIF1 ) antibody . Cell extracts from Tgif1 - / - MEFs cells reconstituted with empty vector ( EV ) , wild - type ( TGIF1 ) or non - phosphorylated form ( TGIF1 . 2TA ) of Flag - TGIF1 were immunoblotted with anti - phospho - TGIF1 or β - Actin as a loading control .

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Specificity of anti-phospho-TGIF1 (pTGIF1) antibody. Cell extracts from Tgif1-/- MEFs cells reconstituted with empty vector (EV), wild-type (TGIF1) or non-phosphorylated form (TGIF1.2TA) of Flag-TGIF1 were immunoblotted with anti-phospho-TGIF1 or β-Actin as a loading control.


Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry . Representative pictures at different stages ( 40x ) are shown .

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Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry. Representative pictures at different stages (40x) are shown.


The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown . Scale bars , 100 μM .

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The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown. Scale bars, 100 μM.


MIAPaCa - 2 cells were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 and resistance colonies were pooled ( n = 6 ) . Expression of Twist1 was examined by qRT - PCR ( D ) .

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MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D).


MIAPaCa - 2 cells were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 and resistance colonies were pooled ( n = 6 ) . Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel .

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MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.


BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1 . 2TA in the presence or absence of KrasG12D . Luciferase activity was measured 48 hr following transfection and normalized on the basis of co - transfected Renilla luciferase ( n = 6 ) .

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BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).


BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1 . 2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in ( E ) ( n = 6 ) .

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BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in (E) (n=6).


Representative microscope pictures ( 20x ) of MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells . Scale bars , 200 μM .

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Representative microscope pictures (20x) of MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells. Scale bars, 200 μM.


Expression of TGIF1 and Twist1 in MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells was analyzed by immunoblotting .

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Expression of TGIF1 and Twist1 in MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells was analyzed by immunoblotting.


MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti - Flag antibody ( C ) .

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MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti-Flag antibody (C).


MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . Cells were selected with neomycin for 2 - 3 weeks , and counted using an automatic cell counter ( D ) ( n = 6 ) .

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MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cells were selected with neomycin for 2-3 weeks, and counted using an automatic cell counter (D) (n=6).


Asynchronously growing human U - 2 OS cells were treated with 0 . 5 Gy of IR , allowed to recover for the indicated time periods , and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC .

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Asynchronously growing human U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC.


Representative images from the QIBC analysis in ( A ) . Scale bars , 10 μm .

Representative images from the QIBC analysis in (A). Scale bars, 10 μm.


Cells were treated with 0 . 5 Gy of IR in absence or presence of 0 . 4 M sorbitol , fixed 1h later , and 53BP1 assembly at DSBs was analyzed by QIBC . Treatments as in ( C ) ; RIF1 accumulation at DSBs was analyzed by QIBC .

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Cells were treated with 0.5 Gy of IR in absence or presence of 0.4 M sorbitol, fixed 1h later, and 53BP1 assembly at DSBs was analyzed by QIBC. Treatments as in (C); RIF1 accumulation at DSBs was analyzed by QIBC.


U - 2 OS cells were treated with 0 . 5 Gy of IR , allowed to recover for the indicated time periods , stained for 53BP1 and MDC1 , and analyzed by QIBC . 53BP1 foci intensities are plotted against MDC1 foci intensities , and representative images are shown .

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U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, stained for 53BP1 and MDC1, and analyzed by QIBC. 53BP1 foci intensities are plotted against MDC1 foci intensities, and representative images are shown.


U - 2 OS cells stably expressing GFP - MDC1 or GFP - 53BP1 were treated with 1 Gy of IR and analyzed by live cell microscopy at 15 min intervals . Representative images are provided and kinetics of GFP - MDC1 and GFP - 53BP1 foci formation and dissolution are shown as single cell tracks . Bold lines represent averages from n = 29 for GFP - MDC1 and n = 18 for GFP - 53BP1 cells .

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U-2 OS cells stably expressing GFP-MDC1 or GFP-53BP1 were treated with 1 Gy of IR and analyzed by live cell microscopy at 15 min intervals. Representative images are provided and kinetics of GFP-MDC1 and GFP-53BP1 foci formation and dissolution are shown as single cell tracks. Bold lines represent averages from n=29 for GFP-MDC1 and n=18 for GFP-53BP1 cells.


U - 2 OS cells were treated with 1 Gy of IR and fixed at the indicated time - points . Mean 53BP1 foci intensity was analyzed from more than 1500 cells per time - point . Mean ( solid line ) and standard deviation from the mean ( dashed lines ) are indicated . The same cells as in ( A ) were analyzed for mean MDC1 foci intensity . Mean ( solid line ) and standard deviation from the mean ( dashed lines ) are indicated .

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U-2 OS cells were treated with 1 Gy of IR and fixed at the indicated time-points. Mean 53BP1 foci intensity was analyzed from more than 1500 cells per time-point. Mean (solid line) and standard deviation from the mean (dashed lines) are indicated. The same cells as in (A) were analyzed for mean MDC1 foci intensity. Mean (solid line) and standard deviation from the mean (dashed lines) are indicated.


GFP - 53BP1 cells were treated with 25 ng / ml NCS to induce DNA breaks and imaged at 30 minutes intervals . Examples of GFP - 53BP1 fusions ( green arrowheads and magnified regions ) and fissions ( blue arrowheads and magnified regions ) are shown .

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GFP-53BP1 cells were treated with 25 ng/ml NCS to induce DNA breaks and imaged at 30 minutes intervals. Examples of GFP-53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown.


53BP1 - RFP cells , in which the endogenous 53BP1 gene locus had been engineered by CRISPR / Cas9 to express 53BP1 - mScarlet from the natural promoter , were treated with NCS ( 25 ng / ml ) and imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads and magnified regions ) and fissions ( blue arrowheads and magnified regions ) are shown on the left and in higher magnification on the right .

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53BP1-RFP cells, in which the endogenous 53BP1 gene locus had been engineered by CRISPR/Cas9 to express 53BP1-mScarlet from the natural promoter, were treated with NCS (25 ng/ml) and imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown on the left and in higher magnification on the right.


GFP - 53BP1 cells were left untreated , or treated with APH ( 0 . 5 μM ) or ATRi ( 1 μM ) to induce replication stress - associated heritable DNA lesions , and cells were imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads and magnified regions ) are shown .

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GFP-53BP1 cells were left untreated, or treated with APH (0.5 μM) or ATRi (1 μM) to induce replication stress-associated heritable DNA lesions, and cells were imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) are shown.


Light - induced optoDroplet formation of Cry2 - mCherry - 53BP1 , Cry2 - mCherry - 53BP1 W1495A , and Cry2 - mCherry - MDC1 . Cells were imaged at 15 seconds intervals . Representative images of optoDroplet formation before and 6 minutes after light induction are shown . Quantifications from single - cell QIBC analysis of 2 - 3 independent experiments are shown with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated .

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Light-induced optoDroplet formation of Cry2-mCherry-53BP1, Cry2-mCherry-53BP1 W1495A, and Cry2-mCherry-MDC1. Cells were imaged at 15 seconds intervals. Representative images of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated.


Cry2 - mCherry - 53BP1 W1495A optoDroplet fusion at 15 seconds time resolution . Fusing optodroplets are highlighted by green arrowheads and in the magnified insets .

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Cry2-mCherry-53BP1 W1495A optoDroplet fusion at 15 seconds time resolution. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.


Light - induced optoDroplet formation of the indicated Cry2 - mCherry - 53BP1 constructs . Cells were imaged at 15 seconds intervals . Representative images and quantifications of optoDroplet formation before and 6 minutes after light induction are shown . Quantifications from single - cell QIBC analysis of 2 - 3 independent experiments are shown with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated . Red bars indicate the part of 53BP1 that was expressed as Cry2 - mCherry fusion .

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Light-induced optoDroplet formation of the indicated Cry2-mCherry-53BP1 constructs. Cells were imaged at 15 seconds intervals. Representative images and quantifications of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion.


Cry2 - mCherry - 53BP1 1203 - 1972 W1495A optoDroplets undergo fusion events upon light induction . Fusing optodroplets are highlighted by green arrowheads and in the magnified insets .

Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets undergo fusion events upon light induction. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.


Concentration - dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2 - mCherry - 53BP1 1203 - 1972 W1495A expression level . Cells were imaged at 15 seconds intervals . Quantifications of optoDroplet formation in lowly expressing cells ( mClow ) and highly expressing cells ( mChigh ) from single - cell QIBC analysis of 3 independent experiments are shown with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated .

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Concentration-dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2-mCherry-53BP1 1203-1972 W1495A expression level. Cells were imaged at 15 seconds intervals. Quantifications of optoDroplet formation in lowly expressing cells (mClow) and highly expressing cells (mChigh) from single-cell QIBC analysis of 3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated.


Cry2 - mEGFP - 53BP1 1203 - 1972 W1495A assembles into FokI - induced DNA repair compartments within seconds after light induction . FokI was induced for 1h where indicated before cells were light - activated and imaged at 15 seconds intervals . Red arrowheads mark the FokI - induced lesion with mCherry accumulation .

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Cry2-mEGFP-53BP1 1203-1972 W1495A assembles into FokI-induced DNA repair compartments within seconds after light induction. FokI was induced for 1h where indicated before cells were light-activated and imaged at 15 seconds intervals. Red arrowheads mark the FokI-induced lesion with mCherry accumulation.