( 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

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(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.


Cry2 - mCherry - 53BP1 1203 - 1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later . Endogenous BRCA1 and p53 were co - stained .

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Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later. Endogenous BRCA1 and p53 were co-stained.


As in ( A ) , comparing Cry2 - mCherry - 53BP1 constructs with and without the BRCT domains . Quantifications of p53 assembly into 53BP1 optoDroplets from single - cell QIBC analysis of 500 - 600 cells per condition are shown below 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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As in (A), comparing Cry2-mCherry-53BP1 constructs with and without the BRCT domains. Quantifications of p53 assembly into 53BP1 optoDroplets from single-cell QIBC analysis of 500-600 cells per condition are shown below 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.


Western blot of p53 induction upon DNA damage ( 10 Gy , 2h ) with and without 0 . 4 M Sorbitol .

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Western blot of p53 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M Sorbitol.


qPCR of p21 induction upon DNA damage ( 10 Gy , 2h ) with and without 0 . 4 M sorbitol . Mean ± standard deviation is indicated .

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qPCR of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. Mean ± standard deviation is indicated.


Western blot of p21 induction upon DNA damage ( 10 Gy , 2h ) with and without 0 . 4 M sorbitol .

Western blot of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol.


QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0 . 4 M sorbitol . Cells were irradiated with 2 Gy in absence or presence of 0 . 4 M sorbitol . After 1h of recovery , sorbitol - containing medium was exchanged with fresh medium , and cells were allowed to recover for an additional 7h . Percentages in G1 and S / G2 are provided .

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QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0.4 M sorbitol. Cells were irradiated with 2 Gy in absence or presence of 0.4 M sorbitol. After 1h of recovery, sorbitol-containing medium was exchanged with fresh medium, and cells were allowed to recover for an additional 7h. Percentages in G1 and S/G2 are provided.


( A ) Cell count - based comparison of cellular proliferation of T - ALL cells after treatment with dexamethasone ( Dexa ) . Individual and mean values of three biological replicates ± SD analyzed by two - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(A) Cell count-based comparison of cellular proliferation of T-ALL cells after treatment with dexamethasone (Dexa). Individual and mean values of three biological replicates ± SD analyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( B ) Flow cytometric analysis of cell death by AnnexinV staining in T - ALL cell lines upon treatment with indicated Dexa concentrations . Cell death is represented by the number of AnnexinV positive ( AnnexinV + ) , apoptotic cells as a percentage ( % ) of total cells . Mean values of n ≥ 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated Dexa concentrations. Cell death is represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells. Mean values of n≥4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( C ) Expression levels of human GRα and LRH - 1 mRNA in human T - ALL cell lines were determined by probe - based real - time quantitative PCR , calculated as relative expression compared to beta - actin and used to determine the GR : LRH - 1 ratio ( right ) . Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM - C7 and GC - resistant cell lines were determined by one - way ANOVA .

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(C) Expression levels of human GRα and LRH-1 mRNA in human T-ALL cell lines were determined by probe-based real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM-C7 and GC-resistant cell lines were determined by one-way ANOVA.


( D ) Immunoblots of GR , BimEL , BimL and BimS and Tubulin from T - ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa . Representative results from n = 2 biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(D) Immunoblots of GR, BimEL, BimL and BimS and Tubulin from T-ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa. Representative results from n=2 biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( E ) Immunoblots of GR , PARP ( nuclear loading control ) and Tubulin ( cytoplasmatic loading control ) from cytoplasmic ( CP ) and nucleoplasmic ( NP ) lysates of T - ALL cells treated with Dexa . Note : Nuclear extracts were more highly concentrated ( approx . 6 - fold ) than cytosolic fractions . Representative results from two biological replicates are shown .

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(E) Immunoblots of GR, PARP (nuclear loading control) and Tubulin (cytoplasmatic loading control) from cytoplasmic (CP) and nucleoplasmic (NP) lysates of T-ALL cells treated with Dexa. Note: Nuclear extracts were more highly concentrated (approx. 6-fold) than cytosolic fractions. Representative results from two biological replicates are shown.


( F ) GR activity in human T - ALL cells transfected with a control luciferase reporter plasmid ( pGL3 ) ( F ) GR responsive ( GRE ) luciferase reporter and treated with 3d2 and / or Dexa . β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO - treated pGL3 control . Individual and mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) GR activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) (F) GR responsive (GRE) luciferase reporter and treated with 3d2 and/or Dexa. β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( G ) LRH - 1 activity in human T - ALL cells transfected with a control luciferase reporter plasmid ( pGL3 ) and ( G ) LRH - 1 responsive ( 5xRE ) luciferase reporter and treated with 3d2 β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO - treated pGL3 control . Individual and mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(G) LRH-1 activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) and (G) LRH-1 responsive (5xRE) luciferase reporter and treated with 3d2 β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( H ) Flow cytometry - based quantification of BiFC in Jurkat cells transiently co - transfected with YFP1 - LRH - 1 and GR - YFP2 and treated for indicated time periods with Dexa . YFP + cells are shown as a percentage ( % ) of viable cells . Individual and mean values ± SD of three biological replicates analyzed by one - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0h ) was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(H) Flow cytometry-based quantification of BiFC in Jurkat cells transiently co-transfected with YFP1-LRH-1 and GR-YFP2 and treated for indicated time periods with Dexa. YFP+ cells are shown as a percentage (%) of viable cells. Individual and mean values ± SD of three biological replicates analyzed by one-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0h) was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( A ) Daily cell counting - based comparison of cellular proliferation of T - ALL cells after ( A ) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(A) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (A) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


( B ) Daily cell counting - based comparison of cellular proliferation of T - ALL cells after ( B ) small hairpin RNA - mediated LRH - 1 knockdown ( shLRH - 1 ) . Individual and mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(B) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (B) small hairpin RNA-mediated LRH-1 knockdown (shLRH-1). Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( C ) Cell cycle distribution of T - ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry . Representative results from n = 3 biological replicates are shown . Stacked bars represent the mean of technical triplicates ± SD . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(C) Cell cycle distribution of T-ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry. Representative results from n=3 biological replicates are shown. Stacked bars represent the mean of technical triplicates ± SD. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( D ) Simultaneous flow cytometric analysis of cell cycle distribution by EdU / DAPI staining and cell death by AnnexinV staining in MOLT - 4 cells upon treatment with 3d2 . Dead cells are represented by the number of AnnexinV positive ( AnnexinV + ) , apoptotic cells as a percentage ( % ) of total cells and cell cycle phase distribution as % of viable , AnnexinV negative ( AnnexinV - ) cells . Representative results from three biological replicates are shown . Bars represent the mean of technical triplicates ± SD and differences in AnnexinV + cells were analyzed by one - way ANOVA . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(D) Simultaneous flow cytometric analysis of cell cycle distribution by EdU/DAPI staining and cell death by AnnexinV staining in MOLT-4 cells upon treatment with 3d2. Dead cells are represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells and cell cycle phase distribution as % of viable, AnnexinV negative (AnnexinV-) cells. Representative results from three biological replicates are shown. Bars represent the mean of technical triplicates ± SD and differences in AnnexinV+ cells were analyzed by one-way ANOVA. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( E ) Flow cytometric analysis of cell death by AnnexinV staining in T - ALL cell lines upon treatment with indicated 3d2 concentrations . Mean values of n = 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(E) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated 3d2 concentrations. Mean values of n=4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( F ) Flow cytometric analysis of 3d2 - induced cell death by AnnexinV staining in wild - type ( WT ) , Bcl - 2 overexpressing ( Bcl - 2 ) and Z - VAD - FMK ( zVAD ) - treated CEM - C1 . Mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD analyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(F) Flow cytometric analysis of 3d2-induced cell death by AnnexinV staining in wild-type (WT), Bcl-2 overexpressing (Bcl-2) and Z-VAD-FMK (zVAD)-treated CEM-C1. Mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD analyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( G ) Immunoblots of full - length and cleaved ( cl . ) PARP , P - p38 , c - Myc , Caspase 3 ( Casp . 3 ) , cl . Casp . 3 , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated with 40 µM 3d2 for indicated time periods . Representative results from two biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(G) Immunoblots of full-length and cleaved (cl.) PARP, P-p38, c-Myc, Caspase 3 (Casp. 3), cl. Casp. 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 for indicated time periods. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


( A ) Immunoblots of full - length and cleaved ( cl . ) PARP , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated for 24 h with 10 µM dexamethasone ( Dexa ) and 3d2 . Representative results from three biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control .

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(A) Immunoblots of full-length and cleaved (cl.) PARP, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated for 24 h with 10 µM dexamethasone (Dexa) and 3d2. Representative results from three biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.


( B ) Flow cytometric analysis of cell death by AnnexinV staining in MOLT - 4 cells treated with 3d2 , 30 µM Dexa and / or 50 µM pan caspase inhibitor Z - VAD - FMK ( zVAD ) . Individual and mean values of technical triplicates of one representative out of n = 3 biological replicates ± SD are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control . Two - way ANOVA ( B ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Flow cytometric analysis of cell death by AnnexinV staining in MOLT-4 cells treated with 3d2, 30 µM Dexa and/or 50 µM pan caspase inhibitor Z-VAD-FMK (zVAD). Individual and mean values of technical triplicates of one representative out of n=3 biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control. Two-way ANOVA (B ns = not significant, ** p < 0.01, *** p < 0.001.


( C ) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout ( Bim KO ) CEM - C7 cells treated with 3d2 and 30 µM Dexa . Mean values of technical triplicates of one representative of n = 4 biological replicates ± SD are shown . Data information : Two - way ANOVA C , ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout (Bim KO) CEM-C7 cells treated with 3d2 and 30 µM Dexa. Mean values of technical triplicates of one representative of n=4 biological replicates ± SD are shown. Data information: Two-way ANOVA C, ns = not significant, ** p < 0.01, *** p < 0.001.