Media from soluble TREM - Fc family member transfected HEK293T incubated with Aβ42 fibrils . Top panel shows original media sample presence of TREM family members associated with Aβ42 pellet . Bottom panel shows original media 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 APOE3 co transfected HEK293T incubated with agarose beads anti - Human IgG Fc . Top panel shows original media sample presence of soluble TREM family members associated with APOE3 . Bottom panel shows original media 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 ) 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 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 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 reverse - incubated with Aβ42 oligomers ( association I ) followed by a second incubation with APOE3 ( association II ) . experiments were replicated 3 independent times

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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 , 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 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 )

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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 , then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM - Fc family members . Results were expressed as 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 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 . No GFP expression was detected with Aβ42 oligomers from control transduced . Results were expressed as ± GFP 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 transduced with TREM2 or variants ( R47H , R62H T96K ) were incubated with 10 µM Aβ42 oligomers for 12 hours . No GFP expression was detected with Aβ42 oligomers from control transduced . Results were expressed as GFP 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 , see Appendix table S3 for p value

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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 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 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 , 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 transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours . Conditioned media lysate were analyzed by Western blot for TREM2 ( RIPA lysate ) 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 transfected with TREM2 were subjected to biotinylation for purification . Bottom panel in D shows presence of TREM2 in purified product . Endogenous APP β - tubulin were used as controls for purification ( middle 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 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 of GLUT1 ( hypoxia marker Control ( n = 5 ) , 2mg ( n = 5 ) , 5mg ( n = 4 ) . In 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 for Glut1 in PDAC tumors assessed by proteomic ( 6 mice per condition 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 ) o CD31 ( endothelial marker ) . Control ( n = 5 ) , 2mg ( n = 5 ) , 5mg ( n = 4 ) . In 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 of DEGs relevant to response to hypoxia ( left ) blood vessel morphogenesis ( right ) . 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 of PDAC tissues from KPC mice treated with vehicle control 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 ) of HIF - 1A in PDAC tissues Control ( n = 5 ) , 5mg ( n = 4 ) . In box - - whisker plot , central box represents values from lower to upper quartile . middle represents mean . vertical extends from minimum to 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 of HIF - 1A in PSCs , normalized to RPLP0 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 expression in PSCs ( p - Tmod is post - translational modification ) . plot shows of sum of band intensities corresponding to isoform 1 , isoform 2 , isoform 3 , post - transcriptionally modified HIF - 1A ( n = 8 control 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 of LOX - L2 in PSCs , normalized to RPLP0 relative to

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


( B ) Western blot 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 - seq data in control 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 of LOX family in PSCs , normalized to RPLP0 relative to

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


( E ) Relative values of for LOX members in PDAC tumors assessed by proteomic ( 6 mice per condition 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 of PDAC tissues from KPC mice treated with vehicle ( control ) , 2mg 5mg of tamoxifen , scale bar 50 μm . ( G ) of LOX - L2 for in F . n ≥ 4 mice per condition , 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 of LOX - L2 HIF - 1A in PSCs , normalized to RPLP0 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 of LOX - L2 HIF - 1A in PSCs , normalized to RPLP0 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 ) of average forces applied by PSCs on pillars . BBI = blebbistatin . In box whisker plot , central box represents values from lower upper quartile . middle represents mean . vertical extends from minimum to 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 of LOX - L2 in PSCs , normalized to RPLP0 ( 60S acidic ribosomal P0 ) 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 ) of matrigel collagen gels previously remodelled by PSCs , second harmonic generation signal for fibrillar collagen ( green ) F - actin ( red ) , scale bar 100 μm . ( B ) Fiber thickness colour - code map in a represented through BoneJ plugin where larger spheres fit along represent greater thickness , scale bar 100 μm . ( C ) SHG fibrillar collagen used for calculation of alignment through FFT algorithm . Insets show FFTs of fibrillar collagen - I , representing alignment with respect to elliptical distribution of FFT central maxima . Circular behaviour ( values approaching 1 ) represent no aligned orientation lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to orientation direction . Scale bar 20 μm ( D - F ) of fiber thickness , , alignment for in A - C . ( G ) of collagen fiber thickness using BoneJ algorithm for in B , H . n ≥ 6 matrices per condition . In scattered plots each point represents a section . In box - - whisker plot , central box represents values from lower to upper quartile . middle represents mean . vertical extends from minimum to maximum value . Histogram bars represent mean ± s . e . m . * * * P < 0 . 001 ( t - for D , E , F ; Anova Tukey " s post hoc for G ) . Three experimental replicates for

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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 of collagen in PSCs , normalized to RPLP0 relative to

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


( B ) Western blot of collagen normalised to total relative to

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


( C ) of time - lapse collagen synthesis deposition by

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


( D ) Representative immunofluorescent used for in C , scale bar 20 μm , collagen - I was assessed with a specific 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 for collagen in PDAC tumors assessed by proteomic ( 6 mice per condition 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 of MMP - 2 in PSCs , normalized to RPLP0 relative to

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


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


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

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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 in PDAC tissues from KPC mice treated with vehicle ( control ) , 2mg 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 for MMP - 2 in PDAC tumors assessed by proteomic ( 6 mice per condition 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 of MMP - 2 in PSCs , normalized to RPLP0 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 of MMP - 2 in PSCs , normalized to RPLP0 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 of fibronectin ( FN ) , fibronectin extracellular domain A ( FN - EDA ) , fibronectin extracellular domain B ( FN - EDB ) in PSCs , normalised to RPLP0 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 of FN normalised to total relative to

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


( C ) of fibronectin density o FN SHG collagen 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 SHG collagen for matrigel collagen gels remodelled by

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


( E ) Immunohistochemistry of PDAC tissues from KPC mice treated with vehicle control , 2mg , 5mg of tamoxifen . ( n = 4 per condition , n ≥ 5 sections per ) . ( F ) FN fiber thickness colour - code map in E represented through 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 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 ) of fibronectin immunohistochemistry , fiber thickness alignment scored of presented in e . In cases , histogram bars represent mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - for A , B , C ; Anova Tukey " s post hoc 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 of PDAC tissues from KPC mice treated with vehicle control , 2mg of tamoxifen , scale bar is 100 μm . Upper panels : Ki67 is used as a surrogate of proliferation . arrows indicate Ki67 positive nuclei in epithelial . Lower panels : Cc3 shows undergoing caspase - mediated apoptosis . Tamoxifen panels show higher of yellow , which indicates higher of apoptotic epithelial . ( B ) of in panel A ( n = 4 per condition , n ≥ 5 sections per , 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 of control tamoxifen treated suit - 2 , scale bar is 20 μm . Panel A represents HIF - 1A in hypoxia non - hypoxia conditions , panels E F show Ki67 Cc3 as markers of proliferation caspase - mediated apoptosis , respectively . Panel E : Red - F - actin , green - Ki67 , blue - nuclei . Tamoxifen negatively regulates HIF - 1A in hypoxia non - hypoxia conditions . ( B , C , D ) of immunofluorescence in panels A , E , F . For , eight fields of view ( n > 50 ) per condition . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t - . 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 3D volumes of humanTFG ( 1 ‐ 193 ; EMDataBank accession code EMD ‐ 6076 ) . Two sets of 2D class averages 3D volumes are shown with left columns showing class averages generated by reference ‐ free alignment classification right columns showing 3D RCT volumes corresponding to class average to 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 3D volumes of C . elegans TFG ( 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 ‐ human TFG truncated TFG ( 1 ‐ 193 ) . truncated form is depicted in yellow , while full ‐ form is depicted as gray mesh . top view ( top ) exhibits limited differences in structures , while side view ( bottom ) shows extra density in full ‐ 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 was determined using dynamic scattering under increasing potassium acetate concentrations . Error bars represent mean ± ; 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 of recombinant , BODIPY ‐ labeled TFG in 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 presence of varying potassium acetate concentrations was imaged by negative stain ‐ EM . Large arrowheads highlight TFG polymers ( only found in presence of elevated potassium acetate ) . Small arrowheads highlight 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 carboxyl ‐ terminus of C . elegans TFG ( 195 ‐ 486 ) . Samples were analyzed at different concentrations , 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 quartz . spectra are characteristic of an intrinsically disordered .

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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 distributions of endogenous TFG ERGIC ‐ 53 were examined in hTERT ‐ RPE ‐ 1 stably expressing low of mApple ‐ Sec16B using SR ‐ SIM ( more than 15 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 stably expressing low of GFP ‐ Sec16B mRuby ‐ Sec23A ( Sec23A ) were fixed 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 distribution of endogenous TFG was examined in stably expressing low 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 distributions of endogenous TFG 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 distributions of endogenous TFG Sec31A were examined using immunogold EM . Large arrowheads highlight 15 nm gold particles bound to α ‐ TFG antibodies . small arrowhead highlights 5 nm gold particles bound to α ‐ Sec31A antibodies . At least 15 different were examined , representative 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 depleted of endogenous TFG were transfected with a variety of TFG transgenes ( including full ‐ , untagged TFG , a truncation mutant encoding 1 ‐ 300 of untagged TFG , a truncation mutant encoding 1 ‐ 350 of untagged TFG ) stained using α ‐ TFG α ‐ Sec31A antibodies . Scale bar , 5 μm . Inset scale bar , 1 μm . shown are representative of at least 15 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 ‐ SUMO ‐ tagged TFG fragment ( Sumo ‐ TFGc , 194 ‐ 400 ; total molecular mass of 42 kDa ) was expressed purified from Escherichia coli extracts using nickel affinity resin . Coomassie ‐ stained gels of peak elution fractions after separation of recombinant on a S200 gel filtration column ( top ) or a 10 - 30 % glycerol gradient ( bottom ) are shown . To determine native molecular of , following equation was used : M = 6πηNas / ( 1 − υρ ) , where M is native molecular , η is viscosity of medium , N is Avogadro ' s number , a is Stokes radius , s is sedimentation value , υ is partial specific volume , ρ is density of 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 ( C ) or depleted of endogenous TFG ( D ) were transfected with a construct expressing TFG with a carboxyl ‐ terminal GFP tag stained using α ‐ Sec31A antibodies . Scale bar , 5 μm . shown are representative of at least 20 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 were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . were fixed stained using α ‐ Sec16A α ‐ Sec31A antibodies imaged using confocal microscopy . shown are projections of 3D data sets ( 4 μm in z ) . Both merged with Sec16A in green Sec31A in red are shown ( at least 15 for each condition ) . Scale bar , 5 μm . Higher magnification views of indicated regions ( boxed ) are also shown in lower or upper right portion of each image . Inset scale bar , 1 μm . B of 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 peri ‐ Golgi were examined . Error bars represent mean ± ; at least 15 different per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ .

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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 of 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 peri ‐ Golgi were examined . Error bars represent mean ± ; at least 15 different per condition . No statistically significant difference was observed , following using a paired t ‐ .

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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 stably expressing low of GFP ‐ ERGIC ‐ 53 mRuby ‐ Sec23A were fixed imaged using SR ‐ SIM ( top ) , or depleted of endogenous TFG prior to fixation SR ‐ SIM imaging ( bottom ) . shown are representative of at least 15 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 were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . were fixed stained using α ‐ Sec16A α ‐ ERGIC ‐ 53 antibodies imaged using SR ‐ SIM . shown are projections of 3D data sets ( 4 μm in z ) . Both merged with Sec16A in green ERGIC ‐ 53 in red are shown ( at least 15 for each condition ) . Scale bar , 5 μm . Higher magnification views of indicated regions ( boxed ) are also shown in lower or upper right portion of each image . Arrowheads highlight Sec16A ‐ labeled structures that do not exhibit juxtaposed ERGIC ‐ 53 . Inset scale bar , 1 μm . F on structured illumination microscopy , of 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 ± ; at least 15 different per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ .

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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 stably expressing low of mannosidase II ‐ mApple ( ManII ‐ mApple ) were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed stained using TFG antibodies imaged using confocal microscopy . Alternatively , 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 . shown are projections of 3D data sets ( 4 μm in z ) . Merged with TFG in green ManII in red are shown . Scale bar , 5 μm . shown are representative of at least 10 analyzed for each condition . B Human RPE ‐ 1 were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed stained using TFG GM130 antibodies imaged using confocal microscopy . Alternatively , 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 . shown are projections of 3D data sets ( 4 μm in z ) . Merged with TFG in green GM130 in red are shown . Scale bar , 5 μm . shown are representative of at least 10 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 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 stably expressing low of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . were subsequently treated with brefeldin A ( BFA ) for 1 h , followed by a wash into fresh media further incubation for 2 h in absence of BFA , prior to fixation . shown are projections of 3D data sets ( 4 μm in z ) . Merged with Sec31A in green ManII in red are shown ( representative of at least 15 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 stably expressing low of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . were subsequently treated with BFA for 1 h , followed by a wash into fresh media further incubation for 1 h in absence of BFA , prior to fixation using α ‐ Sec16A α ‐ Sec31A antibodies . shown are projections of 3D data sets ( 4 μm in z ) . Merged with Sec31A ( green ) , ManII ( red ) , Sec16A ( blue ) are shown ( representative of at least 15 analyzed ) . Scale bar , 5 μm . C Higher magnification views of indicated regions in ( B , boxed ) are shown . Arrows highlight COPII ‐ positive transport carriers that contain cargo ManII , which are not juxtaposed to Sec16A ‐ labeled sites on ER . Additionally , arrowheads point out distinct foci in which COPII continues to associate with Sec16A ‐ labeled sites , indicating that COPII vesicle formation continues in 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 revealed by immunofluorescence ( IF ) 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 eosin ( H & E ) or immunostained with antibodies to CK19 or Amylase revealed by . 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 of control , Tgif1KO , KC KTC mice . A regular mosaic two - colors was used to discriminate between control 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 spleen tissues from control , Tgif1KO , KC or KTC mice ( left ) . of pancreas was measured 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 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 KTC mice . Yellow arrows indicate metastatic nodules in liver . Yellow dotted lines show 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 revealed by IF . Representative pictures at 4x ( Lymph node , H & E ) , 10x ( Liver , H & E ) 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 ) 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).


volume metastasis in live KCLuc KTCLuc mice were analyzed by IVIS bioluminescence . Following imaging live , pancreas , brain , liver lung were dissected analyzed again by IVIS bioluminescence ( n = 6 , 5 out of 6 mice developed metastasis to liver 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 lysates from pancreas of control , KC or KTC mice were pooled 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 ± . * * p < 0 . 01 ; on a two - Student ' s .

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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.


extracts from MIAPaCa - 2 ( left ) control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody 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).


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


extracts from MIAPaCa - 2 ( left ) control were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C , 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 were treated with TGF - β for 30 min , extracts were immunoprecipitated with anti - TGIF1 antibody analyzed by immunoblotting using anti - Twist1 antibody . To control for activation of TGF - β signaling , total 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 - targeting TGIF were treated with vehicle or Dox for 48 hr analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) . In left panel , of bands was assessed by densitometry , result was presented as a 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 analyzed for binding of TGIF1 to Twist1 promoter by ChIP 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 from control or Tgif1KO mice was analyzed for binding of TGIF1 to Twist1 promoter by ChIP 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 Tam - inducible Twist1 ( Twist1ER ) were treated with Tam / or Dox for 48 hr 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 were transfected with wild - type or mutated ( Twist1 binding site ) Twist1 luciferase reporters ( TGTLuc ) together with increasing amounts of Twist1 expression vector in presence of empty vector ( EV ) or TGIF1 expression vector . Luciferase activity was measured 48 hr following transfection normalized on 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 analyzed by immunoblotting using antibodies to E - Cadherin , Vimentin , p16Ink4A 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 , 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 revealed by IF or . 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 of control , KTC 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 revealed by . Representative pictures at 10x ( Liver , H & E ) or 20x ( Pancreas , H & E ; Pancreas , - CK19 ) are shown ( n = 30 ) . Scale bars , 400 μM ( Liver , H & E ) 200 μM ( Pancreas , H & E ; Pancreas , - 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 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 . extracts from Tgif1 - / - MEFs 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 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.


percentages of samples with high versus low expression of pTGIF1 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 were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 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 were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 resistance colonies were pooled ( n = 6 ) . Binding of TGIF1 to Twist1 promoter was examined by ChIP 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 were transfected with TGTLuc together with TGIF1 or TGIF1 . 2TA in presence or absence of KrasG12D . Luciferase activity was measured 48 hr following transfection normalized on 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 were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1 . 2TA in presence or absence of KrasG12D 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 Panc - 1 . 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 Twist1 in MIAPaCa - 2 , Suit - 2 , Capan - 2 Panc - 1 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 Panc - 1 were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . 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 Panc - 1 were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . were selected with neomycin for 2 - 3 , counted using an automatic 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 were treated with 0 . 5 Gy of IR , allowed to recover for indicated time periods , stained for 53BP1 content for cycle resolved of 53BP1 foci in 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 from QIBC in ( A ) . Scale bars , 10 μm .

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


were treated with 0 . 5 Gy of IR in absence or presence of 0 . 4 M sorbitol , fixed 1h later , 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 were treated with 0 . 5 Gy of IR , allowed to recover for indicated time periods , stained for 53BP1 MDC1 , analyzed by QIBC . 53BP1 foci intensities are plotted against MDC1 foci intensities , representative 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 stably expressing GFP - MDC1 or GFP - 53BP1 were treated with 1 Gy of IR analyzed by live microscopy at 15 min intervals . Representative are provided kinetics of GFP - MDC1 GFP - 53BP1 foci formation dissolution are shown as single tracks . Bold lines represent averages from n = 29 for GFP - MDC1 n = 18 for GFP - 53BP1 .

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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 were treated with 1 Gy of IR fixed at indicated time - points . Mean 53BP1 foci was analyzed from more than 1500 per time - point . Mean ( solid ) standard deviation from mean ( dashed lines ) are indicated . same as in ( A ) were analyzed for mean MDC1 foci . Mean ( solid ) standard deviation from 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 were treated with 25 ng / ml NCS to induce breaks imaged at 30 minutes intervals . Examples of GFP - 53BP1 fusions ( green arrowheads magnified regions ) fissions ( blue arrowheads 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 , in which endogenous 53BP1 gene locus had been engineered by CRISPR / Cas9 to express 53BP1 - mScarlet from natural promoter , were treated with NCS ( 25 ng / ml ) imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads magnified regions ) fissions ( blue arrowheads magnified regions ) are shown on left in higher magnification on 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 were left untreated , or treated with APH ( 0 . 5 μM ) or ATRi ( 1 μM ) to induce replication stress - associated heritable lesions , were imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads 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.


- induced optoDroplet formation of Cry2 - mCherry - 53BP1 , Cry2 - mCherry - 53BP1 W1495A , Cry2 - mCherry - MDC1 . were imaged at 15 seconds intervals . Representative of optoDroplet formation before 6 minutes after induction are shown . Quantifications from single - QIBC of 2 - 3 independent experiments are shown with mean ( solid ) standard deviation from 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 in 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.


- induced optoDroplet formation of indicated Cry2 - mCherry - 53BP1 constructs . were imaged at 15 seconds intervals . Representative quantifications of optoDroplet formation before 6 minutes after induction are shown . Quantifications from single - QIBC of 2 - 3 independent experiments are shown with mean ( solid ) standard deviation from mean ( dashed lines ) indicated . Red bars indicate 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 induction . Fusing optodroplets are highlighted by green arrowheads in magnified insets .

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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.


- dependent optoDroplet formation exemplified by two neighboring that differ in their Cry2 - mCherry - 53BP1 1203 - 1972 W1495A expression level . were imaged at 15 seconds intervals . Quantifications of optoDroplet formation in lowly expressing ( mClow ) highly expressing ( mChigh ) from single - QIBC of 3 independent experiments are shown with mean ( solid ) standard deviation from 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 repair compartments within seconds after induction . FokI was induced for 1h where indicated before were - activated imaged at 15 seconds intervals . Red arrowheads mark 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.


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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 without BRCT domains . Quantifications of p53 assembly into 53BP1 optoDroplets from single - QIBC of 500 - 600 per condition are shown below with mean ( solid ) standard deviation from mean ( dashed lines ) indicated . Red bars indicate 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 damage ( 10 Gy , 2h ) with 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 damage ( 10 Gy , 2h ) with 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 damage ( 10 Gy , 2h ) with without 0 . 4 M sorbitol .

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


QIBC cycle on content Cyclin A upon damage with without 0 . 4 M sorbitol . 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 , were allowed to recover for an additional 7h . Percentages in G1 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 ) - comparison of proliferation of T - after treatment with dexamethasone ( Dexa ) . mean values of three biological replicates ± SD analyzed by two - way ANOVA are shown . Data information : For 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 of by AnnexinV in T - lines upon treatment with indicated Dexa concentrations . is represented by number of AnnexinV positive ( AnnexinV + ) , apoptotic as a ( % ) of total . 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 of human GRα LRH - 1 mRNA in human T - lines were determined by probe - real - time quantitative PCR , calculated as relative expression compared to beta - actin used to determine GR : LRH - 1 ( right ) . mean values of four biological replicates ± SD are shown statistically significant differences between CEM - C7 GC - resistant 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 BimS Tubulin from T - treated for 24 h with 20 µM 3d2 or 30 µM Dexa . Representative results from n = 2 biological replicates are shown . Data information : For 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 ( loading control ) Tubulin ( cytoplasmatic loading control ) from ( CP ) nucleoplasmic ( NP ) lysates of T - treated with Dexa . Note : extracts were more highly concentrated ( approx . 6 - fold ) than 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 - transfected with a control luciferase reporter plasmid ( pGL3 ) ( F ) GR responsive ( GRE ) luciferase reporter treated with 3d2 / or Dexa . β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity calculated as relative to DMSO - treated pGL3 control . mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For 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 - transfected with a control luciferase reporter plasmid ( pGL3 ) ( G ) LRH - 1 responsive ( 5xRE ) luciferase reporter treated with 3d2 β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity calculated as relative to DMSO - treated pGL3 control . mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For 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.