A 293T were transfected with plasmids expressing either an empty vector ( Vec ) or vectors expressing HA - tagged STEAP , STAMP1 ( ST1 ) , STAMP2 ( ST2 ) , or STAMP3 ( ST3 ) . Whole - extracts were prepared subjected to Western blot using anti - HA antibody or GAPDH as a loading control .

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A 293T cells were transfected with plasmids expressing either an empty vector (Vec) or vectors expressing HA-tagged STEAP, STAMP1 (ST1), STAMP2 (ST2), or STAMP3 (ST3). Whole-cell extracts were prepared and subjected to Western blot analysis using anti-HA antibody or GAPDH as a loading control.


B Relative ferrireductase activities were measured in expressing different constructs used in ( A ) as described in Materials Methods . * P < 0 . 0001 .

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B Relative ferrireductase activities were measured in the cells expressing the different constructs used in (A) as described in Materials and Methods. *P < 0.0001.


C 293T were transfected with different plasmids expressing either an empty vector ( Vec ) or vectors expressing His - tagged wild - type STAMP2 ( WT ) or three STAMP2 mutants ( dGSR , H304L , or H397L ) as indicated . Whole - extracts were prepared used in Western blot with anti - His antibody or GAPDH antibody as a loading control .

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C 293T cells were transfected with different plasmids expressing either an empty vector (Vec) or vectors expressing His-tagged wild-type STAMP2 (WT) or three STAMP2 mutants (dGSR, H304L, or H397L) as indicated. Whole-cell extracts were prepared and used in Western blot analysis with anti-His antibody or GAPDH antibody as a loading control.


D Relative ferrireductase activities were measured in from ( C ) as described in Materials Methods . * P = 0 . 0003 .

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D Relative ferrireductase activities were measured in the cells from (C) as described in Materials and Methods. *P = 0.0003.


E 293T with Dox - inducible STAMP2 expression or vector control was generated . Western blot confirmed STAMP2 expression in a Dox - inducible manner .

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E 293T cell line with Dox-inducible STAMP2 expression or vector control was generated. Western blot analysis confirmed STAMP2 expression in a Dox-inducible manner.


F from ( E ) were either left untreated or treated with increasing amounts of Dox , ferrireductase activity was determined . * P < 0 . 0001 .

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F The cells from (E) were either left untreated or treated with increasing amounts of Dox, and ferrireductase activity was determined. *P < 0.0001.


G with Dox - inducible STAMP2 expression from ( E ) were either left untreated or treated with Dox for 48 h . Prior to ferrireductase activity measurement , were treated with or without DPI as indicated for 1 h . * P < 0 . 0001 ; * * P = 0 . 0012 ; * * * P = 0 . 048 .

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G The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Prior to ferrireductase activity measurement, the cells were treated with or without DPI as indicated for 1 h. *P < 0.0001; **P = 0.0012; ***P = 0.048.


H from ( E ) were treated with 100 ng / ml Dox for 2 days . Oxidative stress was then measured by NBT , stained were photographed . There were equal numbers of on plates for − / + Dox as shown in Supplementary Fig S4 .

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H The cells from (E) were treated with 100 ng/ml Dox for 2 days. Oxidative stress was then measured by NBT staining, and the stained cells were photographed. There were equal numbers of cells on the plates for −/+ Dox as shown in Supplementary Fig S4.


I with Dox - inducible STAMP2 expression from ( E ) were either left untreated or treated with Dox for 48 h . Then , were treated with or without 10 μM DPI before being subjected to NBT . * P < 0 . 0001 .

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I The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Then, the cells were treated with or without 10 μM DPI before being subjected to NBT staining. *P < 0.0001.


A LNCaP stably expressing an empty vector ( LN - Vec ) or a vector expressing STAMP2 ( LN - ST2 ) were established by lentivirus delivery . lysates were prepared subjected to Western blot with indicated antisera .

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A LNCaP cells stably expressing an empty vector (LN-Vec) or a vector expressing STAMP2 (LN-ST2) were established by lentivirus delivery. Cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.


B Relative ferrireductase activities were measured in described in ( A ) . * P = 0 .

B Relative ferrireductase activities were measured in the cells described in (A). *P = 0.0003


C NBT was performed in LNCaP stably expressing either an empty vector ( LN - Vec ) or a vector expressing wild - type STAMP2 ( LN - ST2 / WT ) or a STAMP2 mutant ( LN - ST2 / dGSR ) as indicated . stained were then photographed quantified . * P = 0 . 004 .

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C NBT staining was performed in LNCaP cells stably expressing either an empty vector (LN-Vec) or a vector expressing wild-type STAMP2 (LN-ST2/WT) or a STAMP2 mutant (LN-ST2/dGSR) as indicated. The stained cells were then photographed and quantified. *P = 0.004.


D LNCaP were transfected with either scrambled siRNA ( siCtrl ) or siRNA against STAMP2 ( siST2 ) were cultured in presence of 10 nM R1881 or vehicle for 2 days . Intracellular ROS were then measured by CellROX reagent . * P = 0 . 002 . ns , not significant .

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D LNCaP cells were transfected with either scrambled siRNA (siCtrl) or siRNA against STAMP2 (siST2) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days. Intracellular ROS levels were then measured by CellROX reagent staining. *P = 0.002. ns, not significant.


E LNCaP stably expressing an empty vector ( Vec ) , a vector expressing wild - type STAMP2 ( WT ) , or a STAMP2 mutant ( dGSR ) were cultured , NADPH / NADP + was determined as described in Materials Methods . * P = 0 . 015 .

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E LNCaP cells stably expressing an empty vector (Vec), a vector expressing wild-type STAMP2 (WT), or a STAMP2 mutant (dGSR) were cultured, and the NADPH/NADP+ ratio was determined as described in Materials and Methods. *P = 0.015.


F LNCaP were transfected with either control siRNA ( siCtrl ) or STAMP2 siRNA ( siST2 ) . were then cultured for 2 days harvested , NADPH / NADP + was determined as above . * P = 0 . 026 .

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F LNCaP cells were transfected with either control siRNA (siCtrl) or STAMP2 siRNA (siST2). Cells were then cultured for 2 days and harvested, and the NADPH/NADP+ ratio was determined as above. *P = 0.026.


G LNCaP stably expressing wild - type STAMP2 were treated with or without DPI ( 1 μM ) for 4 h , harvested , used in NADPH / NADP + . * P = 0 . 018 .

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G LNCaP cells stably expressing wild-type STAMP2 were treated with or without DPI (1 μM) for 4 h, harvested, and used in the NADPH/NADP+ assay. *P = 0.018.


A LNCaP were implanted subcutaneously into nu / nu mice . Once tumors reached 5 mm in , mice ( n = 5 per group ) were given nanoliposome - encapsulated control siRNA or STAMP2 siRNA as described in Materials Methods . volumes were measured at indicated time points . * P = 0 . 001 .

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A LNCaP cells were implanted subcutaneously into nu/nu mice. Once tumors reached 5 mm in size, mice (n = 5 per group) were given nanoliposome-encapsulated control siRNA or STAMP2 siRNA as described in Materials and Methods. Tumor volumes were measured at the indicated time points. *P = 0.001.


B Same experiment as in ( A ) , but VCaP were used instead . * P = 0 . 001 .

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B Same experiment as in (A), but VCaP cells were used instead. *P = 0.001.


( B ) Migration efficiency ( % of that migrated through filter pores in each round ) of A375 during 5 μm constricted migration 12 μm migration ( left panels ) . Biological replicates shown . Phase contrast imaging of final populations after 10 rounds of migration , cultured on 2D plastic ( right panels , Scale bar = 50 µm ) .

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(B) Migration efficiency (% of cells that migrated through filter pores in each round) of A375 cells during 5 μm constricted migration and 12 μm migration (left panels). Biological replicates shown. Phase contrast imaging of the final cell populations after the 10 rounds of migration, cells cultured on 2D plastic (right panels, Scale bar = 50 µm).


( C ) A375 nuclei stained with DAPI ( blue ) , Lamin A / C ( green ) H3K9me3 ( red ) in indicated migration conditions . shown represent a maximum projection of z - stacks ( Scale bar = 5 µm ) .

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(C) A375 nuclei stained with DAPI (blue), Lamin A/C (green) and H3K9me3 (red) in the indicated migration conditions. Images shown represent a maximum projection of z-stacks (Scale bar= 5 µm).


( D ) Cumulative distribution plot of Lamin A / C coefficient of variation in indicated A375 conditions ( Control ( n = 59 ) , Top - 5 ( n = 64 ) , Bottom - 5 ( n = 62 ) nuclei ) . Kolmogorov - Smirnov p - values indicated in plot .

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(D) Cumulative distribution plot of Lamin A/C coefficient of variation in all indicated A375 conditions (Control (n = 59), Top-5 (n = 64), Bottom-5 (n = 62) nuclei). Kolmogorov-Smirnov test p-values indicated in plot.


( E ) Radial distribution of H3K9me3 signal in maximum projected nuclei of Control ( n = 59 ) , Top - 5 ( n = 63 ) , Bottom - 5 ( n = 64 ) Bottom - 12 ( n = 80 ) nuclei ( * * p = 0 . 0069 , two - t - ) . Error bars = mean ± SD .

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(E) Radial intensity distribution of H3K9me3 signal in the maximum projected nuclei of Control (n = 59), Top-5 (n = 63), Bottom-5 (n = 64) and Bottom-12 (n = 80) nuclei (** p = 0.0069, two-tailed t-test). Error bars = mean ± SD.


( F ) Maximum projection confocal of Top - 5 Bottom - 5 embedded in collagen stained with DAPI ( blue ) Phalloidin ( red ) . ( Inset ) Zoom in of nucleus of Bottom - 5 . Top - 5 Bottom - 5 zoomed inset scale = 5 μm ; Bottom - 5 zoomed out image scale = 20 μm .

(F) Maximum projection confocal images of Top-5 and Bottom-5 cells embedded in collagen and stained with DAPI (blue) and Phalloidin (red). (Inset) Zoom in of nucleus of Bottom-5 cell. Top-5 and Bottom-5 zoomed inset scale = 5 μm; Bottom-5 zoomed out image scale = 20 μm.


( G ) Fraction of Control , Top - 5 Bottom - 5 displaying projections at indicated timepoints after were embedded in 3D collagen matrices . Each biological replicate is connected by a .

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(G) Fraction of Control, Top-5 and Bottom-5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices. Each biological replicate is connected by a line.


( H ) Nuclei of Top - 5 ( left ) Bottom - 5 ( right ) embedded in 3D collagen matrix stained with DAPI ( blue ) Lamin A / C ( green ) . Scale = 5μm . For 3D reconstruction visualization , see Movies EV5 EV6 .

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(H) Nuclei of Top-5 (left) and Bottom-5 cells (right) embedded in 3D collagen matrix and stained with DAPI (blue) and Lamin A/C (green). Scale = 5μm. For 3D reconstruction visualization, see Movies EV5 and EV6.


( I ) of embedded in collagen that had elongated nuclei ( Aspect > 2 . 5 ) for A375 Control ( n = 54 ) , Top - 5 ( n = 52 ) Bottom - 5 ( n = 53 ) .

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(I) Percent of cells embedded in collagen that had elongated nuclei (Aspect Ratio > 2.5) for A375 Control (n = 54), Top-5 (n = 52) and Bottom-5 (n = 53).


( J ) Cumulative distribution plot of Lamin A / C coefficient of variation for Control ( n = 70 ) , Top - 5 ( n = 78 ) Bottom - 5 ( n = 109 ) embedded in 3D collagen matrices . Kolmogorov - Smirnov p - values indicated in plot .

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(J) Cumulative distribution plot of Lamin A/C coefficient of variation for Control (n = 70), Top-5 (n = 78) and Bottom-5 (n = 109) cells embedded in 3D collagen matrices. Kolmogorov-Smirnov test p-values indicated in plot.


( E ) Metaphase spread labeled with chromosome specific FISH probes for chr5 ( green ) chr13 ( red ) . Bottom graphs represent frequency of t ( 5 ; 13 ) quantified by FISH ( left ) copy number for chr5 chr13 ( right ) . n = 1328 Control 2370 metaphases analyzed for Bottom - 5 . ( Scale = 10 μm )

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(E) Metaphase spread labeled with chromosome specific FISH probes for chr5 (green) and chr13 (red). Bottom graphs represent frequency of t(5;13) quantified by FISH (left) and copy number quantification for chr5 and chr13 (right). n = 1328 Control and 2370 metaphases analyzed for Bottom-5. (Scale = 10 μm)


The structure of SARS-CoV-2 monomer.


Representative of GINs cultures showing GABA TUJ1 expression . Scale bar = 100 μm .

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Representative images of GINs cultures showing GABA and TUJ1 expression. Scale bar = 100 μm.


proportion of GABA + of TUJ1 + neurons from both CTRL sMDD iPSCs derived GINs at day 35 . ( CTRL , n = 120 fields were counted from 5 lines ; sMDD , n = 134 fields were counted from 6 lines ) .

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The proportion of GABA+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at day 35. (CTRL, n=120 fields were counted from 5 cell lines; sMDD, n=134 fields were counted from 6 cell lines).


Representative schematic morphometric Sholl from CTRL sMDD GINs at day 35 . Sholl intersection number of CTRL sMDD GINs at day 35 ( Each red box is from 5 CTRL lines , n = 25 independently differentiations from 5 lines , each point represents mean value of neurons from five independent experiments , n = 226 neurons from CTRL groups . Each blue box is from 6 sMDD lines , n = 30 differentiations independently from 6 lines , each point represents mean value of neurons from five independent experiments , n = 260 neurons from sMDD groups ) . Two - way ANOVA for different from soma , * * * * p ＜ 0 . 0001 for 10 , 30 50 μm .

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Representative images and schematic morphometric Sholl analysis from CTRL and sMDD GINs at day 35. Sholl intersection number of CTRL and sMDD GINs at day 35 (Each red box is from 5 CTRL cell lines, n=25 independently differentiations from 5 cell lines, each point represents mean value of neurons from five independent experiments, n=226 neurons from CTRL groups. Each blue box is from 6 sMDD cell lines, n=30 differentiations independently from 6 cell lines, each point represents mean value of neurons from five independent experiments, n=260 neurons from sMDD groups). Two-way ANOVA for different distance from soma, ****p＜0.0001 for 10, 30 and 50 μm.


Representative of GINs from CTRL sMDD at day 65 .

Representative images of GINs from CTRL and sMDD at day 65.


of branch numbers of GINs shown in 5 CTRL lines 6 sMDD lines at day 65 , n ≥ 11 , Nested t - , * * p = 0 . 0016 for CTRL versus sMDD . of longest neurite of GINs shown in 5 CTRL lines 6 sMDD lines at day 65 , n ≥ 11 , Nested t - , * * p = 0 . 0075 for CTRL versus sMDD .

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Quantification of primary branch numbers of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65, n≥11, Nested t-test, **p=0.0016 for CTRL versus sMDD. Quantification of longest neurite length of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65, n≥11, Nested t-test, **p=0.0075 for CTRL versus sMDD.


Representative of GINs subtype calretinin ( CR ) expression . proportion of CR + of TUJ1 + neurons from both CTRL sMDD iPSCs derived GINs at days 35 , 40 50 , respectively . ( Red bar from 5 CTRL lines , blue bar from 6 sMDD lines ) . n = 5 in each . Two - way ANOVA for timepoints ,

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Representative images of GINs subtype calretinin (CR) expression. The proportion of CR+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at days 35, 40 and 50, respectively. (Red bar from 5 CTRL cell lines, blue bar from 6 sMDD cell lines). n=5 in each cell line. Two-way ANOVA for timepoints,


Representative of GINs subtype somatostatin ( SST ) expression co - with mature neuron marker MAP2 in CTRL sMDD GINs at day 65 . proportion of SST + neurons of MAP2 + from CTRL sMDD iPSCs derived GINs at day 65 . n = 43 in CTRL groups n = 37 in sMDD groups .

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Representative images of GINs subtype somatostatin (SST) expression co-staining with mature neuron marker MAP2 in CTRL and sMDD GINs at day 65. The proportion of SST+ neurons of MAP2+ cells from CTRL and sMDD iPSCs derived GINs at day 65. n=43 in CTRL groups and n=37 in sMDD groups.


Representative electrophysiological traces of AP at a holding potential of - 70 mV from GINs in sMDD ( blue ) CTRL ( red ) groups . Average number of APs evoked during 500ms stepwise depolarization ( CTRL , n = 35 neurons from 3 lines ; sMDD , n = 55 neurons from 3 lines ) .

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Representative electrophysiological traces of AP at a holding potential of -70 mV from GINs in sMDD (blue) and CTRL (red) groups. Average number of APs evoked during 500ms stepwise depolarization (CTRL, n=35 neurons from 3 lines; sMDD, n=55 neurons from 3 lines).


D - F . Amplitude half width of first AP generated in response to a 10 - pA injection ( CTRL , n = 35 neurons from 3 lines ; sMDD , n = 55 neurons from 3 lines ) . Nested t - , * * p ＜ 0 . 01 ; * * * p ＜ 0 . 001 . Mean ± .

D-F. Amplitude and half width of the first AP generated in response to a 10-pA injection (CTRL, n=35 neurons from 3 lines; sMDD, n=55 neurons from 3 lines). Nested t-test, **p＜0.01; ***p＜0.001. Mean ratio ± SEM.


G . Traces of Na + / K + currents recorded from GINs in sMDD CTRL groups . showed average of peak values of Na + currents ( CTRL , n = 46 neurons from 3 lines ; sMDD , n = 38 neurons from 3 lines ) . Nested t - , * * p ＜ 0 . 01 . H . Average fast potassium currents of GINs in sMDD CTRL groups ( CTRL , n = 43 neurons from 3 lines ; sMDD , n = 36 neurons from 3 lines ) .

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G. Traces of Na+/K+ currents recorded from GINs in sMDD and CTRL groups. Quantification showed average of peak values of Na+ currents (CTRL, n=46 neurons from 3 lines; sMDD, n=38 neurons from 3 lines). Nested t-test, **p＜0.01. H. Average fast potassium currents of GINs in sMDD and CTRL groups (CTRL, n=43 neurons from 3 lines; sMDD, n=36 neurons from 3 lines).


Representative of calcium imaging in CTRL sMDD GINs at each timepoint . Scale bar = 20 μm .

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Representative images of calcium imaging in CTRL and sMDD GINs at each timepoint. Scale bar = 20 μm.


representative trajectory of average changes over time from CTRL ( red ) sMDD ( blue ) neurons in response to 67 mM KCL . CTRL , n = 6 neurons ; sMDD , n = 6 neurons . Traces are from a representative experiment ( whole result is shown in D ) . of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per ( n = 155 neurons derived from 5 CTRL lines , n = 198 neurons derived from 6 sMDD lines ) . Nested t - , * * p = 0 . 0022 for CTRL versus sMDD .

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The representative trajectory of average intensity changes over time from CTRL (red line) and sMDD (blue line) neurons in response to 67 mM KCL. CTRL, n=6 neurons; sMDD, n=6 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=155 neurons derived from 5 CTRL cell lines, n=198 neurons derived from 6 sMDD cell lines). Nested t-test, **p=0.0022 for CTRL versus sMDD.


Immunostaining for GABA interneuron marker GAD67 , neural progenitor marker SOX2 ventral prosencephalic progenitor marker NKX2 . 1 at day 30 in CTRL sMDD ventral forebrain organoids .

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Immunostaining for the GABA interneuron marker GAD67, neural progenitor marker SOX2 and ventral prosencephalic progenitor marker NKX2.1 at day 30 in CTRL and sMDD ventral forebrain organoids.


Representative of calcium imaging in CTRL sMDD ventral forebrain organoids at different states .

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Representative images of calcium imaging in CTRL and sMDD ventral forebrain organoids at different states.


of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per ( n = 156 neurons derived from 4 CTRL lines , n = 88 neurons derived from 4 sMDD lines ) . Nested t - , * * * p = 0 . 0005 for CTRL versus sMDD .

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Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=156 neurons derived from 4 CTRL cell lines, n=88 neurons derived from 4 sMDD cell lines). Nested t-test, ***p=0.0005 for CTRL versus sMDD.


Bar plot showing number of genes associated with known mental illnesses , on PsyGeNET . " Total " presents of genes related to a given disease . " 100 % association " presents genes only positively correlated with disease . " 100 % no association " presents genes only negatively correlated with disease . " Both " means genes that both positive negative results were found . AUD , BD , CUD , MDD SCZ represent alcohol use disorder , bipolar disorder , cocaine use disorder , major depressive disorder schizophrenia , respectively .

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Bar plot showing the number of genes associated with known mental illnesses, based on PsyGeNET. "Total" presents all of the genes related to a given disease. "100% association" presents the genes only positively correlated with the disease. "100% no association" presents the genes only negatively correlated with the disease. "Both" means genes that both positive and negative results were found. AUD, BD, CUD, MDD and SCZ represent alcohol use disorder, bipolar disorder, cocaine use disorder, major depressive disorder and schizophrenia, respectively.


illustrating expression level of DEGs enriched in selected Reactome pathways , gene ontology ( GO ) terms , Kyoto Encyclopedia of Genes Genomes ( KEGG ) pathways , which is normalized by row .

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Heatmap illustrating the expression level of DEGs enriched in selected Reactome pathways, gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which is normalized by row.


Calcium imaging was performed on GFP + neurons in knockdown group . representative image shows state of GFP + state of after KCl stimulation . pseudo - color showed Rhod - 4 calcium dye , selected are GFP + neurons .

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Calcium imaging was performed on GFP+ neurons in the knockdown group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons.


representative trajectory of average changes over time in Vehicle KD groups . CTRL , n = 10 neurons ; sMDD , n = 10 neurons . Traces are from a representative experiment ( whole result is shown in I ) . of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per group . n = 89 for Vehicle , n = 118 for KD , respectively from 4 batches . T - , * * * * p < 0 . 0001 .

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The representative trajectory of average intensity changes over time in Vehicle virus and KD virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in I). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=89 for Vehicle virus, n=118 for KD virus, respectively from 4 batches. T-test, ****p<0.0001.


Calcium imaging was performed on GFP + neurons in overexpression group . representative image shows state of GFP + state of after KCl stimulation . pseudo - color showed Rhod - 4 calcium dye , selected are GFP + neurons .

Calcium imaging was performed on GFP+ neurons in the overexpression group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons.


representative trajectory of average changes over time in Vehicle OE groups . CTRL , n = 10 neurons ; sMDD , n = 10 neurons . Traces are from a representative experiment ( whole result is shown in D ) . of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per group . n = 166 for Vehicle , n = 81 for OE , respectively from 3 batches .

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The representative trajectory of average intensity changes over time in Vehicle virus and OE virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=166 for Vehicle virus, n=81 for OE virus, respectively from 3 batches.


Representative of calcium imaging in CTRL ( from RC01001 - C ) sMDD ( from SA004 ) GINs at resting stimulus state with different treatments .

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Representative images of calcium imaging in CTRL (from RC01001-C) and sMDD (from SA004) GINs at resting and stimulus state with different treatments.


of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown with different treatments including BDNF , Trzd , PNU , Ro at concentrations of 1 μM 10 μM . T - , * * p = 0 . 0042 for sMDD + BDNF versus sMDD ; one - way ANOVA , * * * * p ＜ 0 . 0001 for sMDD + 10 μM Trzd versus sMDD . n ≥ 7 neurons from each group .

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Quantification of peak [Ca2+] (Fmax-F0)/F0 shown with different treatments including BDNF, Trzd, PNU, Ro at concentrations of 1 μM and 10 μM. T-test, **p=0.0042 for sMDD+BDNF versus sMDD; one-way ANOVA, ****p＜0.0001 for sMDD+10 μM Trzd versus sMDD. n≥7 neurons from each group.


Dot plots showing expression level of genes selected from functional gene sets across CTRL , sMDD sMDD with Trzd GINs clusters .

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Dot plots showing the expression level and percentage of genes selected from functional gene sets across CTRL, sMDD and sMDD with Trzd GINs clusters.


Top : Violin plots showing expression level of CAMK2N1 CAMK2N2 in three groups . Bottom : Scatter plot showing correlations of foldchanges according to expression of genes encoding CaMK family between CTRL GINs sMDD GINs between sMDD with Trzd GINs sMDD GINs . A linear regression is added to plot on these two variables . sMDD + Trzd vs . sMDD , n = 14 genes ; CTRL vs . sMDD , n = 14 genes .

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Top: Violin plots showing the expression level of CAMK2N1 and CAMK2N2 in three groups. Bottom: Scatter plot showing the correlations of foldchanges according to the expression of genes encoding CaMK family proteins between CTRL GINs and sMDD GINs and between sMDD with Trzd GINs and sMDD GINs. A linear regression line is added to the plot based on these two variables. sMDD+Trzd vs. sMDD, n=14 genes; CTRL vs. sMDD, n=14 genes.


Representative of single neuron from CTRL , CTRL + Trzd , sMDD sMDD + Trzd GINs .

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Representative images of single neuron from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs.


Representative chart of Sholl intersection number over from soma in CTRL , CTRL + Trzd , sMDD sMDD + Trzd GINs at day 45 . CTRL , n = 81 neurons from 5 lines ; CTRL + Trzd , n = 81 neurons from 5 lines ; sMDD , n = 73 neurons from 6 lines ; sMDD + Trzd , n = 71 neurons from 6 lines . of maximum intersections from CTRL , CTRL + Trzd , sMDD sMDD + Trzd GINs via Sholl . CTRL , n = 81 neurons from 5 lines ; CTRL + Trzd , n = 81 neurons from 5 lines ; sMDD , n = 73 neurons from 6 lines ; sMDD + Trzd , n = 71 neurons from 6 lines .

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Representative line chart of Sholl intersection number over distance from soma in CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs at day 45. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines. Quantification of maximum intersections from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs via Sholl analysis. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines.


of sum of intersections from CTRL , CTRL + Trzd , sMDD sMDD + Trzd GINs via Sholl . CTRL , n = 81 neurons from 5 lines ; CTRL + Trzd , n = 81 neurons from 5 lines ; sMDD , n = 73 neurons from 6 lines ; sMDD + Trzd , n = 71 neurons from 6 lines . One - way ANONA , * * * * p ＜ 0 . 0001 for CTRL versus sMDD ; * * * p = 0 . 0002 for sMDD versus sMDD + Trzd . representative trajectory of average changes over time from CTRL , CTRL + Trzd , sMDD sMDD + Trzd GINs in response to 67 mM KCL . CTRL , n = 20 neurons ; CTRL + Trzd , n = 20 neurons ; sMDD , n = 20 neurons ; sMDD + Trzd , n = 21 neurons . Tracs are from a representative experiment ( whole result is shown in Fig EV5E ) .

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Quantification of sum of intersections from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs via Sholl analysis. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines. One-way ANONA, ****p＜0.0001 for CTRL versus sMDD; ***p=0.0002 for sMDD versus sMDD+Trzd. The representative trajectory of average intensity changes over time from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs in response to 67 mM KCL. CTRL, n=20 neurons; CTRL+Trzd, n=20 neurons; sMDD, n=20 neurons; sMDD+Trzd, n=21 neurons. Tracs are from a representative experiment (the whole quantification result is shown in Fig EV5E).


of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per group , CTRL , CTRL + Trzd , sMDD sMDD + Trzd from 3 lines . T - , * p = 0 . 0495 for CTRL versus sMDD , * * p = 0 . 0075 for sMDD versus sMDD + Trzd .

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Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per group, CTRL, CTRL+Trzd, sMDD and sMDD+Trzd all from 3 cell lines. T-test, *p=0.0495 for CTRL versus sMDD, **p=0.0075 for sMDD versus sMDD+Trzd.


Representative of calcium imaging in sMDD sMDD + Trzd ventral forebrain organoids at different states .

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Representative images of calcium imaging in sMDD and sMDD+Trzd ventral forebrain organoids at different states.


of effect of Trzd on peak [ Ca2 + ] ( Fmax - F0 ) / F0 in ventral forebrain organoids ( CTRL from IMR90 - 4 , n = 40 ; CTRL + Trzd from IMR90 - 4 , n = 58 ; sMDD from SA007 , n = 49 ; sMDD + Trzd from SA007 , n = 64 ) . Two - , * * * * p ＜ 0 . 0001 for CTRL versus sMDD ; * * * p = 0 . 0007 for sMDD versus sMDD + Trzd .

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Quantification of the effect of Trzd on peak [Ca2+] (Fmax-F0)/F0 in ventral forebrain organoids (CTRL from IMR90-4, n=40 cells; CTRL+Trzd from IMR90-4, n=58 cells; sMDD from SA007, n=49 cells; sMDD+Trzd from SA007, n=64 cells). Two-tailed test, ****p＜0.0001 for CTRL versus sMDD; ***p=0.0007 for sMDD versus sMDD+Trzd.


Representative electrophysiological traces of AP at a holding potential of - 70 mV from GINs in sMDD sMDD + Trzd groups . Average number of APs evoked during 500ms stepwise depolarization ( CTRL , n = 35 neurons from 3 lines ; CTRL + Trzd , n = 30 neurons from 3 lines ; sMDD , n = 55 neurons from 3 lines ; sMDD + Trzd , n = 44 neurons from 3 lines ) .

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Representative electrophysiological traces of AP at a holding potential of -70 mV from GINs in sMDD and sMDD+Trzd groups. Average number of APs evoked during 500ms stepwise depolarization (CTRL, n=35 neurons from 3 lines; CTRL+Trzd, n=30 neurons from 3 lines; sMDD, n=55 neurons from 3 lines; sMDD+Trzd, n=44 neurons from 3 lines).


Traces of Na + / K + currents were recorded from GINs in sMDD sMDD + Trzd groups . Average peak values of Na + currents ( CTRL , n = 46 neurons from 3 lines ; CTRL + Trzd , n = 32 neurons ; sMDD , n = 38 neurons from 3 lines ; sMDD + Trzd , n = 36 neurons from 3 lines ) .

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Traces of Na+/K+ currents were recorded from GINs in sMDD and sMDD+Trzd groups. Average peak values of Na+ currents (CTRL, n=46 neurons from 3 lines; CTRL+Trzd, n=32 neurons; sMDD, n=38 neurons from 3 lines; sMDD+Trzd, n=36 neurons from 3 lines).


of mitotic defects as in ( B ) of H2B - mNeon - expressing HeLaEGFP - AID - CENATAC treated as indicated ( three or five biological replicates , > 85 in total per condition ) . For 2xZF four zinc - finger cysteines were mutated to alanines ; for Δ1 - 4 corresponding motif was removed .

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Quantification of mitotic defects as in (B) of H2B-mNeon-expressing HeLaEGFP-AID-CENATAC cells treated as indicated (three or five biological replicates, >85 cells in total per condition). For 2xZF the four zinc-finger cysteines were mutated to alanines; for Δ1-4 the corresponding motif was removed.


Representative stills of HeLaEGFP - AID - CENATAC expressing H2B - mNeon depleted of CENATAC . Microtubules were visualized with SiR - Tubulin . Arrowheads arrows indicate non - congressed chromosomes supernumerary spindle poles , respectively . Scale bar , 5 μm . Time in hours . See Figure EV2 for control condition . See also Movies EV1 EV2 . IAA , 3 - indoleacetic acid .

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Representative stills of HeLaEGFP-AID-CENATAC cells expressing H2B-mNeon and depleted of CENATAC. Microtubules were visualized with SiR-Tubulin. Arrowheads and arrows indicate non-congressed chromosomes and supernumerary spindle poles, respectively. Scale bar, 5 μm. Time in hours. See Figure EV2 for the control condition. See also Movies EV1 and EV2. IAA, 3-indoleacetic acid.


Graph of fold - changes of enriched ( P - value < 0 . 05 ) in proteomics of CENATAC vs . control co - immunoprecipitations of HeLaEGFP - CENATAC ( three biological replicates ) . Splicing factors are depicted in orange . See also Appendix Figure S7 .

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Graph of fold-changes of proteins enriched (P-value < 0.05) in proteomics analysis of CENATAC vs. control co-immunoprecipitations of HeLaEGFP-CENATAC cells (three biological replicates). Splicing factors are depicted in orange. See also Appendix Figure S7.


Glycerol gradient ( 10 - 30 % ) of HeLa S3 extracts . snRNAs were detected by Northern blot , ( CENATAC PRPF4 ) by western blot . Locations of U6atac mono - snRNP , U4atac / U6atac di - snRNP U4atac / U6atac . U5 tri - snRNP are indicated .

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Glycerol gradient (10-30%) analysis of HeLa S3 nuclear extracts. snRNAs were detected by Northern blot analysis, proteins (CENATAC and PRPF4) by western blot. Locations of the U6atac mono-snRNP, U4atac/U6atac di-snRNP and U4atac/U6atac.U5 tri-snRNP are indicated.


RT - PCRs of IPO5 , SUDS3 ZCCHC8 minor adjacent major introns on extracted from lymphoblasts or HeLaEGFP - AID - CENATAC depleted of GAPDH or CENATAC as indicated . u , unspliced ; s , spliced .

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RT-PCRs of IPO5, SUDS3 and ZCCHC8 minor and adjacent major introns on RNA extracted from patient lymphoblasts or HeLaEGFP-AID-CENATAC cells depleted of GAPDH or CENATAC as indicated. u, unspliced; s, spliced.


RT - PCRs of ZRSR2 GAPDH ( to visualize ZRSR2 knockdown efficiency , upper ) RT - PCRs of SUDS3 minor intron 7 ( middle ) major intron 5 ( lower ) on extracted from HeLaEGFP - AID - CENATAC or DLD - 1 depleted of GAPDH or ZRSR2 as indicated .

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RT-PCRs of ZRSR2 and GAPDH (to visualize ZRSR2 knockdown efficiency, upper) and RT-PCRs of SUDS3 minor intron 7 (middle) and major intron 5 (lower) on RNA extracted from HeLaEGFP-AID-CENATAC or DLD-1 cells depleted of GAPDH or ZRSR2 as indicated.


RT - PCR of major ( U2 - type ) AT - AC introns in SYCP2 ( intron 5 ) TAF2 ( intron 1 ) , minor ( U12 - type ) AT - AC intron in IPO5 ( intron 21 ) on extracted from HeLaEGFP - AID - CENATAC depleted of GAPDH or CENATAC . Schematic representations of unspliced / spliced PCR products are depicted on right .

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RT-PCR of major (U2-type) AT-AC introns in SYCP2 (intron 5) and TAF2 (intron 1), and minor (U12-type) AT-AC intron in IPO5 (intron 21) on RNA extracted from HeLaEGFP-AID-CENATAC cells depleted of GAPDH or CENATAC. Schematic representations of unspliced/spliced PCR products are depicted on the right.


RT - PCR P120 reporter ( Hall & Padgett , 1996 ) to measure relative usage of A - type ( AT - AC ) G - type ( GT - AG ) 5 ' splice sites in direct competition . Upper : schematic diagram showing overall architecture of reporter construct with its down - upstream splice site ( thick red blue bars , respectively ) products created by splicing ( Dn Up , respectively ) . Lower : RT - PCRs of reporter with A - or G - type splice sites in down - or upstream positions as indicated below gel . SS , splice site . * , PCR product after use of a cryptic major splice site ( not shown in schematic ) .

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RT-PCR P120 reporter assay(Hall & Padgett, 1996) to measure the relative usage of A-type (AT-AC) and G-type (GT-AG) 5' splice sites in direct competition. Upper: schematic diagram showing the overall architecture of the reporter construct with its down- and upstream splice site (thick red and blue bars, respectively) and the products created by splicing (Dn and Up, respectively). Lower: RT-PCRs of the reporter with A- or G-type splice sites in the down- or upstream positions as indicated below the gel. SS, splice site. *, PCR product after use of a cryptic major splice site (not shown in the schematic).


A . Immunoblots of subcellular fractions from HeLa exposed to normoxia . P : post - supernatant ; C : cytosol ; ER : endoplasmic reticulum ; MT : mitochondria ; MAM : mitochondrial - associated membranes . B . Immunoblots of subcellular fractions from HeLa exposed to hypoxia ( 1 % O2 ) for 5 h . P : post - supernatant ; C : cytosol ; ER : endoplasmic reticulum ; MT : mitochondria ; MAM : mitochondrial - associated membranes . C . of MAM : PNS of FUNDC1 or DRP1 under normoxic or hypoxic conditions . quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 .

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A. Immunoblots of subcellular fractions from HeLa cells exposed to normoxia. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.B. Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia (1% O2) for 5 h. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.C. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ** P < 0.01.


D . Control HeLa or hypoxia - treated HeLa ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) . Bar = 500 nm . boxed areas in left panels are magnified in right panels . Arrows indicate gold particles .

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D. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Arrows indicate gold particles.


E . Control HeLa or hypoxia - treated HeLa ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - TOM20 antibody ( 12 nm gold particles ) . Bar = 500 nm .

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E. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-TOM20 antibody (12 nm gold particles). Bar = 500 nm.


F . of MAM : mitochondrion of FUNDC1 immunogold particles from D under normoxic hypoxic ( 1 % O2 ) conditions ; Data ( mean ± s . e . m . ) are from 3 independent experiments . n = 60 MAMs ( one MAM means one mitochondrial ER tethering site in corresponding mitochondria ) , * * P < 0 . 01 .

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F. Quantification of the MAM:mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic (1% O2) conditions; Data (mean ± s.e.m.) are from 3 independent experiments. n = 60 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria), ** P < 0.01.


G . of MAM : mitochondrion of TOM20 immunogold particles from E under normoxic hypoxic ( 1 % O2 ) conditions . Data ( mean ± s . e . m . ) are from 3 independent experiments . n = 50 MAMs ( one MAM means one mitochondrial ER tethering site in corresponding mitochondria ) . NS means not significant .

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G. Quantification of the MAM:mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic (1% O2) conditions. Data (mean ± s.e.m.) are from 3 independent experiments. n = 50 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria). NS means not significant.


H. The quantification of MAM counts from 50 cells, ***P < 0.001.


I . of FACL4 immunogold particles from 50 , * * * P < 0 . 001 .

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I. The quantification of FACL4 immunogold particles from 50 cells, *** P < 0.001.


J . Hypoxia - treated HeLa ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) anti - FACL4 ( 6 nm gold particles ) . Bar = 500 nm . boxed areas in left panels are magnified in right panels . Yellow arrows indicate FUNDC1 - gold particles green arrows indicate FACL4 - gold particles .

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J. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-FACL4 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate FACL4-gold particles.


K . of FACL4 immunogold particles on MAM mitochondrion . n = 80 MAMs ( one MAM means one mitochondrial ER tethering site in corresponding mitochondria ) .

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K. The ratio of FACL4 immunogold particles on the MAM and mitochondrion. n = 80 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria).


L . Hypoxia - treated HeLa ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) anti - DRP1 ( 6 nm gold particles ) . Bar = 500 nm . boxed areas in left panels are magnified in right panels . Yellow arrows indicate FUNDC1 - gold particles green arrows indicate DRP1 - gold particles .

L. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-DRP1 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate DRP1-gold particles.


B . HeLa were transfected with FUNDC1 - MYC or empty vector for 24 h . lysates were immunoprecipitated by anti - MYC antibody immunoblotted with indicated antibodies .

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B. HeLa cells were transfected with FUNDC1-MYC or empty vector for 24 h. Cell lysates were immunoprecipitated by anti-MYC antibody and immunoblotted with indicated antibodies.


C . HeLa were transfected with FLAG - FUNDC1 . About 24 h post - transfection , lysates were immunoprecipitated by anti - Calnexin immunoblotted with anti - FLAG anti - Calnexin antibodies .

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C. HeLa cells were transfected with FLAG-FUNDC1. About 24 h post-transfection, cell lysates were immunoprecipitated by anti-Calnexin and immunoblotted with anti-FLAG and anti-Calnexin antibodies.


D E . HeLa were transfected with indicated FUNDC1 - MYC constructs for 24 h . lysates were immunoprecipitated using anti - Calnexin immunoblotted using anti - MYC anti - Calnexin antibodies .

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D and E. HeLa cells were transfected with the indicated FUNDC1-MYC constructs for 24 h. Cell lysates were immunoprecipitated using anti-Calnexin and immunoblotted using anti-MYC and anti-Calnexin antibodies.


F . Immunoblots of subcellular fractions from HeLa transfected with scramble siRNA exposed to hypoxia ( 1 % O2 ) for 5 h . PNS : post - supernatant ; CYTO : cytosol ; ER : endoplasmic reticulum ; MITO : mitochondria ; MAM : mitochondrial - associated . DRP1 ( S ) DRP1 ( L ) indicate short long exposures , respectively . G . Immunoblots of subcellular fractions from HeLa transfected with Calnexin siRNA ( si - CNX ) exposed to hypoxia ( 1 % O2 ) for 5 h . PNS : post supernatant ; CYTO : cytosol ; ER : endoplasmic reticulum ; MITO : mitochondria ; MAM : mitochondrial - associated . DRP1 ( S ) DRP1 ( L ) indicate short long exposures , respectively . H . of MAM : PNS of FUNDC1 or DRP1 in HeLa treated with scramble siRNA or si - CNX exposed to hypoxia ( 1 % O2 ) for 5 h . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 .

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F. Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia (1% O2) for 5 h. PNS: post-nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.G. Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA (si-CNX) and exposed to hypoxia (1% O2) for 5 h. PNS: post nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.H. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si-CNX and exposed to hypoxia (1% O2) for 5 h. Data are presented as mean ± s.e.m. from 3 independent experiments; *** P < 0.001.


A . HeLa were transfected with indicated si - RNAs . 24 h post - transfection , were exposed to hypoxia ( 1 % O2 ) for 12 h then immunostained by anti - TOM20 . Bar = 50 µm . Boxed regions in left panels are enlarged in right panels . C . of mitochondria ( fragmented , intermediate elongated mitochondria ) in 100 from ( A ) . Data represent mean ± s . e . m . ( * * * P < 0 . 001 ) .

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A. HeLa cells were transfected with the indicated si-RNAs. 24 h post-transfection, cells were exposed to hypoxia (1% O2) for 12 h and then immunostained by anti-TOM20. Bar = 50 µm. Boxed regions in the left panels are enlarged in the right panels.C. Quantification of mitochondria morphology (fragmented, intermediate and elongated mitochondria) in 100 cells from (A). Data represent mean ± s.e.m. (*** P<0.001).


B . Electron microscopy of HeLa treated with indicated si - RNAs for 24 h then exposed to hypoxia ( 1 % O2 ) for 12 h . Bar = 1 μm . Boxed regions in left panels are enlarged in right panels .

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B. Electron microscopy analysis of HeLa cells treated with the indicated si-RNAs for 24 h and then exposed to hypoxia (1% O2) for 12 h. Bar = 1 μm. Boxed regions in the left panels are enlarged in the right panels.


A . HeLa were treated with indicated si - RNAs exposed to hypoxia for 12 h . were fixed immunostained by anti - TOM20 anti - DRP1 antibodies . Bar = 40 µm .

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A. HeLa cells were treated with the indicated si-RNAs and exposed to hypoxia for 12 h. Cells were fixed and immunostained by anti-TOM20 and anti-DRP1 antibodies. Bar = 40 µm.


B . Immunogold electron microscopy of control HeLa ( top ) or HeLa transfected with si - FUNDC1 ( bottom ) then exposed to hypoxia ( 1 % O2 ) for 12 h . DRP1 was detected with anti - DRP1 ( 12 nm gold particles ) . Blue stars indicate mitochondria . Yellow arrows indicate gold particles . Red arrows indicate double - membraned autophagosomes .

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B. Immunogold electron microscopy of control HeLa cells (top) or HeLa cells transfected with si-FUNDC1 (bottom) and then exposed to hypoxia (1% O2) for 12 h. DRP1 was detected with anti-DRP1 (12 nm gold particles). Blue stars indicate mitochondria. Yellow arrows indicate gold particles. Red arrows indicate double-membraned autophagosomes.


C - H . Immunoblots of subcellular fractions from control HeLa or HeLa treated with indicated si - then exposed to hypoxia for 5 h . PNS : post supernatant ; Cyto : cytosol ; Mito ( crude ) : crude mitochondrial fraction containing mitochondria MAMs .

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C-H. Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si-RNA and then exposed to hypoxia for 5 h. PNS: post nuclear supernatant; Cyto: cytosol; Mito (crude): crude mitochondrial fraction containing mitochondria and MAMs.


A . HeLa were co - transfected with FLAG - DRP1 FUNDC1 - MYC for 24 h . were lysed immunoprecipitated using anti - FLAG antibody . FLAG - DRP1 FUNDC1 - MYC were analyzed with corresponding antibodies .

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A. HeLa cells were co-transfected with FLAG-DRP1 and FUNDC1-MYC for 24 h. Cells were lysed and immunoprecipitated using anti-FLAG antibody. FLAG-DRP1 and FUNDC1-MYC were analyzed with the corresponding antibodies.


B . HeLa were co - transfected with empty vector , MYC - DRP1 FLAG - FUNDC1 . 24 h after transfection , lysates were immunoprecipitated using anti - FLAG antibody immunoblotted with anti - MYC anti - FLAG antibodies .

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B. HeLa cells were co-transfected with empty vector, MYC-DRP1 and FLAG-FUNDC1. 24 h after transfection, cell lysates were immunoprecipitated using anti-FLAG antibody and immunoblotted with anti-MYC and anti-FLAG antibodies.


C . MEFs were transfected with FUNDC1 - MYC . 24 h post - transfection , were fixed immunostained by anti - MYC anti - DRP1 . Bar = 10 µm .

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C. MEFs were transfected with FUNDC1-MYC. 24 h post-transfection, cells were fixed and immunostained by anti-MYC and anti-DRP1. Bar = 10 µm.


D E . HeLa were co - transfected with indicated FUNDC1 - MYC FLAG - DRP1 constructs for 24 h . lysates were analyzed by immunoprecipitation using anti - FLAG immunoblotted with anti - MYC anti - FLAG antibodies .

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D and E. HeLa cells were co-transfected with the indicated FUNDC1-MYC and FLAG-DRP1 constructs for 24 h. Cell lysates were analyzed by immunoprecipitation using anti-FLAG and immunoblotted with anti-MYC and anti-FLAG antibodies.


F . HeLa were exposed to hypoxia ( 1 % O2 ) for indicated times lysates were immunoprecipitated using anti - FUNDC1 antibody . Endogenous DRP1 , Calnexin , LC3 FUNDC1 were detected by anti - DRP1 , anti - Calnexin , anti - LC3 , anti - FUNDC1 antibodies .

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F. HeLa cells were exposed to hypoxia (1% O2) for the indicated times and cell lysates were immunoprecipitated using anti-FUNDC1 antibody. Endogenous DRP1, Calnexin, LC3 and FUNDC1 were detected by anti-DRP1, anti-Calnexin, anti-LC3, and anti-FUNDC1 antibodies.


G . 10 - 20 g GST - FUNDC1 or GST was immobilized on glutathione sepharose resin ( GE Healthcare ) . 20 - 50 g DRP1 was incubated with GST - FUNDC1 or GST bound resin overnight at 4 ° C . resin was then extensively rinsed eluted . Samples were subjected to SDS - PAGE , then visualized by coomassie blue western blot , respectively .

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G. 10-20 g GST-FUNDC1 or GST was immobilized on glutathione sepharose resin (GE Healthcare). 20-50 g DRP1 was incubated with the GST-FUNDC1 or GST bound resin overnight at 4 °C. The resin was then extensively rinsed and eluted. Samples were subjected to SDS-PAGE, and then visualized by coomassie blue staining and western blot, respectively.


H . Full - FUNDC1 - MYC , FUNDC1 - MYC ( AA 96 - 155 ) , FL ( Δ129 - 138 ) or vector control were transfected with Mito - DsRed ( red ) into FUNDC1 KD MEFs . 12 h post - transfection , were exposed to hypoxia for 12 h then stained with anti - MYC ( blue ) anti - DRP1 ( green ) . Bar = 10 µm . Numbers at right show average of mitochondria in Mito - DsRed - positive .

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H. Full-length FUNDC1-MYC, FUNDC1-MYC (AA 96-155), FL (Δ129-138) or vector control were transfected with Mito-DsRed (red) into FUNDC1 KD MEFs. 12 h post- transfection, cells were exposed to hypoxia for 12 h and then stained with anti-MYC (blue) and anti-DRP1 (green). Bar = 10 µm. Numbers at the right show the average length of mitochondria in Mito-DsRed-positive cells.


A . HeLa were transfected with indicated si - RNAs for 24 h then exposed to hypoxia for 24 h or not . were fixed immunostained by anti - TOM20 anti - LC3 antibodies . colocalization of LC3 mitochondria was quantified listed on right side . Bar = 5 µm .

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A. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h or not. Cells were fixed and immunostained by anti-TOM20 and anti-LC3 antibodies. The colocalization of LC3 and mitochondria was quantified and listed on the right side. Bar = 5 µm.


B C . HeLa were transfected with indicated si - RNAs for 24 h then exposed to hypoxia for 24 h . were then treated with or without 50 nM BAF A1 ( Bafilomycin A1 ) for 6 h before harvesting . lysates were immunoblotted with indicated antibodies .

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B and C. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h. Cells were then treated with or without 50 nM BAF A1 (Bafilomycin A1) for 6 h before harvesting. Cell lysates were immunoblotted with the indicated antibodies.


D E . of TIM23 : Actin TOM20 : Actin ratios from ( B C ) . data are from 3 independent experiments .

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D and E. Quantification of the TIM23:Actin and TOM20:Actin ratios from (B and C). All data are from 3 independent experiments.


F . HeLa were transfected by different truncated FUNDC1 - MYC . About 24h post - transfection , lysates were immunoblotted using indicated anti - bodies .

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F. HeLa cells were transfected by different truncated FUNDC1-MYC. About 24h post-transfection, cell lysates were immunoblotted using indicated anti-bodies.


( A ) Relative mRNA of FX in CD45 + leukocytes in peripheral blood of E0771 ( abbreviated to E ) or LLC tumour - bearing mice . mean sizes of E0771 LLC tumours were 9 . 8 mm 9 . 5 mm , respectively . Shown are averages ( N = 8 mice / group ) with one - way ANOVA

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(A) Relative mRNA levels of FX in CD45+ leukocytes in the peripheral blood of E0771 (abbreviated to E) or LLC tumour-bearing mice. The mean sizes of E0771 and LLC tumours were 9.8 mm and 9.5 mm, respectively. Shown are averages (N = 8 mice/group) with SEM and one-way ANOVA


( B ) Relative mRNA of FX in CD45 + leukocytes in various organs such as lung ( Lu ) , Liver ( Li ) , spleen ( Sp ) , bone marrow ( BM ) lymph node ( Lymph ) derived from no tumour - bearing or E0771 - bearing mice . " Tumour " stands for mRNA of FX in E0771 tumours . Shown are averages ( N = 6 : organs from no tumour - bearing mice , N = 6 : organs from E0771 - bearing mice ) with one - way

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(B) Relative mRNA levels of FX in CD45+ leukocytes in various organs such as lung (Lu), Liver (Li), spleen (Sp), bone marrow (BM) and lymph node (Lymph) derived from no tumour-bearing or E0771-bearing mice. "Tumour" stands for mRNA levels of FX in E0771 tumours. Shown are averages (N = 6: organs from no tumour-bearing mice, N = 6: organs from E0771-bearing mice) with SEM and one-way ANOVA


( C ) Immunohistochemical quantifications of FX expression in CD45 + leukocytes in liver lungs of no tumour - bearing or E0771 - bearing mice . Shown are averages ( N = 6 ) with Welch " s t -

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(C) Immunohistochemical quantifications of FX expression in CD45+ leukocytes in the liver and lungs of no tumour-bearing or E0771-bearing mice. Shown are averages (N = 6) with SEM and Welch"s t-test


( D ) Immunohistochemical quantifications of fibrinogen deposition in no tumour - bearing - tumour - bearing - mouse lungs . Shown are averages ( N = 7 / group , 14 random fields ) with . Welch " s t -

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(D) Immunohistochemical quantifications of fibrinogen deposition in no tumour-bearing- and tumour-bearing- mouse lungs. Shown are averages (N = 7/ group, 14 random fields) with SEM. Welch"s t-test


( E ) Representative photos of immunohistochemical co - localisation of FX + CD45 + fibrinogen deposition in tumour - bearing mouse lungs . Circles show high fibrinogen deposition areas ( scale bar , 50 μm )

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(E) Representative photos of immunohistochemical co-localisation of FX+CD45+ cells and fibrinogen deposition in tumour-bearing mouse lungs. Circles show high fibrinogen deposition areas (scale bar, 50 μm)


( B ) KikGR red were detected in TCM - stimulated - liver lungs after liver exposure to violet ( arrow ) . taken from with no exposure were also shown ( scale bar , 100 μm )

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(B) KikGR red cells were detected in the TCM-stimulated- liver and lungs after liver exposure to violet light (arrow). Images taken from animals with no light exposure were also shown (scale bar, 100 μm)


( C ) Flow cytometric quantifications of photoconverted HepELs in TCM ( 3 times ) - stimulated KikGR mouseliver lungs . were isolated 72 h after photoconversion . was calculated as number of photoconverted ( KikGR redin comparison with number of liver or lung pre - sorted with CD45 - beads . Shown are averages ( N = 5 ) with Welch " s t -

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(C) Flow cytometric quantifications of photoconverted HepELs in TCM (3 times)-stimulated KikGR mouseliver and lungs. Cells were isolated 72 h after photoconversion. Ratio was calculated as the number of photoconverted cells (KikGR redin comparison with the number of liver or lung cells pre-sorted with CD45-beads. Shown are averages (N = 5) with SEM and Welch"s t-test


( D ) Surface marker analyses of photoconverted KikGR . Vertical axes represent of each marker + / photoconverted KikGR . Shown are averages ( conCM , control : N = 4 , TCM : N = 9 ) with Welch " s t -

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(D) Surface marker analyses of photoconverted KikGR cells. Vertical axes represent ratio of each marker+ cell/photoconverted KikGR cell. Shown are averages (conCM, control: N = 4, TCM: N = 9) with SEM and Welch"s t-test


( E ) Representative immunostaining of FX expression in NK1 . 1 + , CD11c + , B220 + CD4 + in tumour - bearing lungs ( scale bar , 10 μm )

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(E) Representative immunostaining images of FX expression in NK1.1+, CD11c+, B220+ and CD4+ cells in tumour-bearing lungs (scale bar, 10 μm)


( A ) Flow cytometric of B220 + CD11c + NK1 . 1 + in lung of TCM - stimulated mice . Values in dot plots present ratios for total gated KikGR red KikGR green

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(A) Flow cytometric analysis of B220+CD11c+NK1.1+ cells in the lung of TCM-stimulated mice. Values in the dot plots present ratios for total cells gated KikGR red and KikGR green cell


( B ) Representative flow cytometric analyses of relocation of B220 + CD11c + NK1 . 1 + in lungs that were photoconverted in TCM - stimulated KikGR mouse liver ( gated in red polygonal region ) . is shown for total ( upper panel in B ) , for gated KikGR red or KikGR green ( lower panels in B ) . Region where photoconverted locate is magnified in right . Yellow arrow shows photoconversion direction . Three independent experiments . LTCM stands for TCM derived from LLC

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(B) Representative flow cytometric analyses of the relocation of B220+CD11c+NK1.1+ cells in lungs that were photoconverted in TCM-stimulated KikGR mouse liver (gated in red polygonal region). Percent is shown for total cells (upper panel in B), and for gated KikGR red or KikGR green cells (lower panels in B). Region where the photoconverted cells locate is magnified in the right. Yellow arrow shows the photoconversion direction. Three independent experiments. LTCM stands for TCM derived from LLC


( A ) Representative photos of IFNγ expression in B220 + CD11c + NK1 . 1 + derived from TCM - stimulating mouse liver . These were cultured with LiCM or LuCM that had been prepared by culture with tumour - bearing - liver or lungs , respectively . NoCM stands for CM without tissues , it was used as control ( scale bar , 10 μm )

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(A) Representative photos of IFNγ expression in B220+CD11c+NK1.1+cells derived from TCM-stimulating mouse liver. These cells were cultured with LiCM or LuCM that had been prepared by culture with tumour-bearing- liver or lungs, respectively. NoCM stands for CM without tissues, and it was used as control (scale bar,10 μm)


( B ) Immunohistochemical quantifications of IFNγ in B220 + CD11c + NK1 . 1 + . derived from liver spleen were primed with LiCM or LuCM . Shown are averages ( N = 6 / group ) with one - way ANOVA

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(B) Immunohistochemical quantifications of IFNγ in B220+CD11c+NK1.1+ cells. Cells derived from liver and spleen were primed with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( C ) Flow cytometric of living Zombie - tumour with B220 + CD11c + NK1 . 1 + that were primed by NoCM or LuCM . NoCM : N = 3 , LuCM : N = 4 . Shown are averages with . Welch " s t - . Rhodamin + tumour were counted as viable

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(C) Flow cytometric analysis of living Zombie- tumour cells with B220+CD11c+NK1.1+ cells that were primed by NoCM or LuCM. NoCM: N=3, LuCM: N=4. Shown are averages with SEM. Welch"s t-test. Rhodamin+ tumour cells were counted as viable cells


( D ) Representative immunohistochemical stainings of rhodamin + ( red ) Zombie + ( green ) tumour ( scale bar , 10 μm )

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(D) Representative immunohistochemical stainings of rhodamin+ (red) Zombie+ (green) tumour cells (scale bar, 10 μm)


( E ) Ratios of living tumour after co - culture with B220 + CD11c + NK1 . 1 + that had been primed with lung - or liver - CM . Shown are averages ( N = 6 / group ) with one - way ANOVA

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(E) Ratios of living tumour cells after co-culture with B220+CD11c+NK1.1+cells that had been primed with lung- or liver-CM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( A ) Western blotting of fibrinogen in tumour - bearing no tumour - bearing livers

(A) Western blotting of fibrinogen in tumour-bearing and no tumour-bearing livers


( C ) IP - Western blot shows - interactions between fibrinogen Vtn / TSP

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(C) IP-Western blot analysis shows protein-protein interactions between fibrinogen and Vtn/TSP


( D ) of fibrinogen deposition areas after injection of CD45 + derived from liver in tumour - bearing mice . Shown are averages ( N = 7 / group ) with . 1 - way ANOVA

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(D) Quantification of fibrinogen deposition areas after injection of CD45+ cells derived from the liver in tumour-bearing mice. Shown are averages (N = 7/ group) with SEM. 1-way ANOVA


( E ) Quantifications of fibrinogen deposition areas in TCM - stimulating wild - type mouse lungs after injection of Vtn + HepELs . Vtn + were isolated from E0771 - bearing wild - type or FX - / - mouse liver by using anti - Vtn beads ( 1 x 106 / mouse ) . Shown are averages ( N = 5 / group , 3 fields / sample ) with . Welch " s t -

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(E) Quantifications of fibrinogen deposition areas in TCM-stimulating wild-type mouse lungs after injection of Vtn+ HepELs. Vtn+ cells were isolated from E0771-bearing wild-type or FX-/-mouse liver by using anti-Vtn beads (1 x 106 cells /mouse). Shown are averages (N=5/ group, 3 fields/ sample) with SEM. Welch"s t-test


( F ) Separation of Vtn + / CD45 + HepELs from tumour - bearing FX + / - FX - / - mouse livers by a sorter ( Moflo Astrios ) . Sorted Vtn + CD45 + HepELs obtained from a littermate ( 3 each ) were pooled , 8 x 103 Vtn + CD45 + HepELs were i . v . injected into a tumour - bearing mouse . Right graph shows fibrinogen depositions in lung . Shown are averages ( N = 3 / group , 9 fields / sample ) with . 1 - way

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(F) Separation of Vtn+/ CD45+ HepELs from tumour-bearing FX+/- and FX-/- mouse livers by a cell sorter (Moflo Astrios). Sorted Vtn+CD45+HepELs cells obtained from a littermate (3 animals each) were pooled, and 8 x 103 Vtn+CD45+HepELs cells were i.v. injected into a tumour-bearing mouse. Right graph shows fibrinogen depositions in the lung. Shown are averages (N = 3/ group, 9 fields/ sample) with SEM. 1-way ANOVA


( A ) Representative immunostaining of fibrinogen depositions in tumour - bearing mouse lungs received an injection of rhodamine - labelled B220 + CD11c + NK1 . 1 + HepELs derived from tumour - bearing - FX + / − or FX − / − mice ( scale bar , 20 μm )

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(A) Representative immunostaining of fibrinogen depositions in tumour-bearing mouse lungs received an injection of rhodamine-labelled B220+CD11c+NK1.1+HepELs derived from tumour-bearing- FX+/− or FX−/− mice (scale bar, 20 μm)


( B ) Immunohistochemical quantifications of fibrinogen depositions in tumour - bearing mouse lung after an injection of B220 + CD11c + NK1 . 1 + derived from tumour - bearing - FX + / − or FX − / − mouse liver . Sorted B220 + CD11c + NK1 . 1 + were prepared from three mice per each genotype , were i . v . injected . Shown are averages ( N = 6 / group , 4 sections / mouse ) with Welch " s t -

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(B) Immunohistochemical quantifications of fibrinogen depositions in tumour-bearing mouse lung after an injection of B220+CD11c+NK1.1+cells derived from tumour-bearing- FX+/− or FX−/− mouse liver. Sorted B220+CD11c+NK1.1+cells were prepared from three mice per each genotype, and were i.v. injected. Shown are averages (N = 6/group, 4 sections/mouse) with SEM and Welch"s t-test


( B ) Relative mRNA of FX , Vtn TSP1 in liver B220 + CD11c + NK1 . 1 + stimulated with LiCM or LuCM . Shown are averages ( N = 6 / group ) with one - way ANOVA

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(B) Relative mRNA levels of FX, Vtn and TSP1 in liver B220+CD11c+NK1.1+cells stimulated with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( C ) Relative mRNA of FX in liver B220 + CD11c + NK1 . 1 + stimulated with various factors . Shown are averages ( N = 4 ) with one - way ANOVA

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(C) Relative mRNA levels of FX in liver B220+CD11c+NK1.1+cells stimulated with various factors. Shown are averages (N = 4) with SEM and one-way ANOVA


( D ) In vitro adhesion . Liver B220 + CD11c + NK1 . 1 + were seeded on a fibrinogen plate . attached to plate were counted . Twentyμg / mL of anti - Vtn Ab or isotype control IgG were used to block adhesion . Shown are averages ( N = 6 / group ) with one - way ANOVA

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(D) In vitro adhesion assay. Liver B220+CD11c+NK1.1+cells were seeded on a fibrinogen plate. Cells attached to the plate were counted. Twentyμg/mL of anti-Vtn Ab or isotype control IgG were used to block the adhesion. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( E ) Lung B220 + CD11c + NK1 . 1 + were seeded on ECM coated ( fibrinogen , fibronectin , or collagen I ) plates . Shown are averages ( N = 5 / group ) with one - way ANOVA

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(E) Lung B220+CD11c+NK1.1+cells were seeded on ECM coated (fibrinogen, fibronectin, or collagen I) plates. Shown are averages (N = 5/group) with SEM and one-way ANOVA


( B ) Tumour homing in lungs pre - treated with LiCM - primed B220 + CD11c + NK1 . 1 + from tumour - bearing WT or FX − / − mouse liver . Representative photos of homing of rhodamine - labelled tumour ( upper , scale bar , 20 μm ) . Number of homing tumour are shown ( lower ) . Shown are averages ( N = 60 sections , 5 / group , field / section , 12 sections / sample ) with Welch " s t -

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(B) Tumour cell homing in the lungs pre-treated with LiCM-primed B220+CD11c+NK1.1+cells from tumour-bearing WT or FX−/− mouse liver. Representative photos of homing of rhodamine-labelled tumour cells (upper, scale bar, 20 μm). Number of homing tumour cells are shown (lower). Shown are averages (N = 60 sections, 5/group, all field count/section, 12 sections/sample) with SEM and Welch"s t-test


( C ) Tumour homing in lungs pre - treated with activated FX - overexpressed B220 + CD11c + NK1 . 1 + ( FX - OE - HepELs ) from tumour - bearing lungs . Number of homing tumour after an injection of lung HepELs or FX - OE - HepELs are shown . Shown here are averages ( N = 24 sections , 4 / group , field / section , 6 sections / sample ) one - way ANOVA

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(C) Tumour cell homing in the lungs pre-treated with activated FX-overexpressed B220+CD11c+NK1.1+cells (FX-OE-HepELs) from tumour-bearing lungs. Number of homing tumour cells after an injection of lung HepELs or FX-OE-HepELs are shown. Shown here are averages (N = 24 sections, 4/group, all field count/section, 6 sections/sample) SEM and one-way ANOVA


( A - B ) Representative Hematoxylin Eosin ( H & E ) , immunohistochemistry of phosphorylated FAK on tyrosine ( Y ) 397 ( pY397 FAK ) , alpha - Smooth Muscle Actin ( α - SMA ) , Fibroblast Activated alpha ( FAP - α ) in serial sections of human normal pancreas ( Ct1 ) , of human normal adjacent PDAC tissue ( Ct2 ) of human PDAC tissues ( PDAC1 - 3 ) ; scale bar , 100 μm ( A ) , 300 µm ( B ) .

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(A-B) Representative Hematoxylin and Eosin (H&E) staining, and immunohistochemistry of phosphorylated FAK on tyrosine (Y) 397 (pY397 FAK), alpha-Smooth Muscle Actin (α-SMA), and Fibroblast Activated Protein alpha (FAP-α) in serial sections of human normal pancreas (Ct1), of human normal adjacent PDAC tissue (Ct2) and of human PDAC tissues (PDAC1-3); scale bar, 100 μm (A), and 300 µm (B).


( C ) of pY397 FAK level in cancer - associated fibroblasts from human PDAC microarray : 40 different PDAC samples 10 normal human pancreas have been stained for pY397 FAK double blinded quantified ( on positive number ) on a scale between 0 ( no ) 6 ( Y397 FAK highly positive ) in whole ( left ) , cytoplasm ( middle ) nucleus ( right ) .

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(C) Quantification of pY397 FAK level in cancer-associated fibroblasts from human PDAC microarray: 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified (based on intensity and positive cell number) on a scale between 0 (no staining) and 6 (Y397 FAK highly positive) in whole cell (left), cytoplasm (middle) and nucleus (right).


( A ) Representative of Definiens ® Tissue studio multi - resolution segmentation algorithm on two PDAC TMA examples ( X , Y ) . From left to right : for pY397 FAK ; ROI detection of tumour ( blue ) , stroma ( orange ) other ( blue / grey ) ; / cytoplasm detection in blue green respectively ; classification for pY397 FAK signal ( red for high signal , orange for medium signal , yellow for low signal for negative signal ) .

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(A) Representative images of Definiens® Tissue studio multi-resolution segmentation algorithm on two PDAC TMA examples (X, Y). From left to right: IHC for pY397 FAK; ROI detection of tumour cells (dark blue), stroma (orange) and other (light blue/grey); Nuclear/ cytoplasm detection in blue and green respectively; IHC staining classification for pY397 FAK intensity signal (dark red for high signal, orange for medium signal, yellow for low signal and white for negative signal).


( B - C ) Disease Free Survival ( B ) Overall Survival ( C ) from 120 cohort according to pY397 FAK expression in CAFs ; HR : Hazard . Survival curves were estimated with Kaplan - Meier method compared using log - rank , non - parametric ( chi2 - ) to compare independent groups for categorical data .

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(B-C) Disease Free Survival (B) and Overall Survival (C) from 120 patient cohort according to pY397 FAK expression in CAFs; HR: Hazard Ratio. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.


( D - E ) Disease Free Survival ( D ) Overall Survival ( E ) from 120 cohort according to pY397 FAK expression in tumour . Survival curves were estimated with Kaplan - Meier method compared using log - rank , non - parametric ( chi2 - ) to compare independent groups for categorical data .

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(D-E) Disease Free Survival (D) and Overall Survival (E) from 120 patient cohort according to pY397 FAK expression in tumour cells. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.


Tumour were syngeneically orthotopically co - grafted with mouse FAK - WT or FAK - KD fibroblasts . Thirty - eight days after injection , mice were euthanized . ( A ) tumour mass . Values are means ± from at least 7 mice per group .

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Tumour cells were syngeneically and orthotopically co-grafted with mouse FAK-WT or FAK-KD fibroblasts. Thirty-eight days after injection, mice were euthanized. (A) Primary tumour mass quantification. Values are means ± SEM from at least 7 mice per group.


( B ) Representative from tumour stained for H & E , cytokeratin 19 ( CK19 ) , α - SMA , Sirius Red polarized Sirius Red . Scale bar , 100 μm .

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(B) Representative images from primary tumour stained for H&E, cytokeratin 19 (CK19), α-SMA, Sirius Red and polarized Sirius Red. Scale bar, 100 μm.


( C ) of fibrillar collagen I ( Yellow - Orange ) III ( Green ) on polarized Sirius Red from B . Each dot represents a mouse ( n = 11 per group ) .

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(C) Quantification of fibrillar collagen I (Yellow-Orange) and III (Green) based on polarized Sirius Red images from B. Each dot represents a mouse (n=11 per group).


representative ( left ) ( right ) expression of CD31 ( D ) from tumour ; scale bar , 100 μm .

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IHC representative images (left) and quantification (right) the expression of CD31 (D) from primary tumour; scale bar, 100 μm.


representative ( left ) ( right ) expression of two hypoxia markers CaIX HIF1α ( E ) from tumour ; scale bar , 100 μm .

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IHC representative images (left) and quantification (right) the expression of two hypoxia markers CaIX and HIF1α (E) from primary tumour; scale bar, 100 μm.


( F ) Representative of H & E , CK19 α - SMA performed on lung sections . Arrow identified lung metastasis . ( G ) of lung metastasis number on CK19 positive using NanoZoomer software . Values are means ± of 6 mice per group .

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(F) Representative images of H&E, CK19 and α-SMA IHC staining performed on lung sections. Arrow identified lung metastasis. (G) Quantification of lung metastasis size and number based on CK19 positive staining using NanoZoomer software. Values are means ± SEM of 6 mice per group.


( A - B ) Relative frequencies of tumour - infiltrating M1 macrophages M2 macrophages analyzed by flow cytometry at 21 days ( A ) 38 days ( B ) after grafting .

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(A-B) Relative frequencies of tumour-infiltrating M1 macrophages and M2 macrophages analyzed by flow cytometry at 21 days (A) and 38 days (B) after grafting.


( D ) Impact of conditioned media ( CM ) secreted by activated mouse fibroblasts expressing FAK - WT or FAK - KD on mouse BMDM - derived macrophage polarization . Relative frequencies of macrophages expressing CD80 , CD86 , Nos2 , CD206 , CMH2 , Dectin , CD163 markers acquired by flow cytometry processed using FlowJo software .

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(D) Impact of conditioned media (CM) secreted by activated mouse fibroblasts expressing FAK-WT or FAK-KD on mouse BMDM-derived macrophage polarization. Relative frequencies of macrophages expressing CD80, CD86, Nos2, CD206, CMH2, Dectin, and CD163 markers acquired by flow cytometry and processed using FlowJo software.


( E ) Impact of soluble factors secreted by CAFs on macrophage migration . Left : schematic of BMDM derived M1 or M2 - like macrophages . Right : Representative pictures of M1 M2 macrophage migration at 24 48h towards CM secreted by CAFs ( previously treated or not with FAK inhibitor for 48h ) . were stained using Cristal Violet of cristal violet positive quantified using Observer videomicroscope ( Zeiss ) analyzed with ImageJ .

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(E) Impact of soluble factors secreted by CAFs on macrophage migration. Left: schematic of BMDM derived M1 or M2-like macrophages. Right: Representative pictures and quantification of M1 and M2 macrophage migration at 24 and 48h towards CM secreted by CAFs (previously treated or not with FAK inhibitor for 48h). Cells were stained using Cristal Violet and percentage of cristal violet positive area quantified using Cell Observer videomicroscope (Zeiss) and analyzed with ImageJ.


( F ) Quantitative real - time PCR conducted with gene - specific primers ( CCL2 , CCL5 M - CSF ) on four different FAK - I - treated ( 16h ) non - treated CAFs for gene expression . normalized fold change ± was calculated using delta - delta Ct method with RSP16 as a control gene ; values are mean + / - . Each - derived activated fibroblasts was assigned to a specific

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(F) Quantitative real-time PCR conducted with gene-specific primers (CCL2, CCL5 and M-CSF) on four different FAK-I-treated (16h) and non-treated CAFs for gene expression. The normalized fold change ± SEM was calculated using the delta-delta Ct method with RSP16 as a control gene; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol


( G ) Elisa of CCL2 ( MCP1 ) ( µg / ml ) in conditioned medium from hCAFs treated or not with FAK - I during 72h . * p < 0 . 05 , by paired t - ; values are mean + / - . Each - derived activated fibroblasts was assigned to a specific

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(G) Elisa quantification of CCL2 (MCP1) concentration (µg/ml) in conditioned medium from hCAFs treated or not with FAK-I during 72h. * p <0.05, by paired t-test; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol


( H ) Left : Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine ( Y ) 397 of CD206 in serial sections of human PDAC tissues . Scale bar : 200µm Middle : Correlation plot between fibroblastic pY397 FAK score CD45 or CD206 + number within human PDAC samples . Fibroblastic pY397 FAK score of CD206 positive quantifications were performed on a 47 cohort on . Spearman r = 0 . 4145 , p = 0 . 0019 . Right : of CD206 positive in PDAC expressing either low or high fibroblastic FAK activity score .

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(H) Left: Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine (Y) 397 and of CD206 in serial sections of human PDAC tissues. Scale bar: 200µm Middle: Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206+ cell number within human PDAC samples. Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining. Spearman r = 0.4145, p=0.0019. Right: Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score.


( A ) Representative of three independent scratch wound assays of co - cultivated red - labelled pancreatic tumour with either FAK - WT ( wild - type ) or FAK - KD ( Kinase - dead ) green - labelled fibroblasts at time zero 46 hours ( corresponding to wound closure of co - cultured FAK - WT fibroblasts with tumour ) .

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(A) Representative images of three independent scratch wound assays of co-cultivated red-labelled pancreatic tumour cells with either FAK-WT (wild-type) or FAK-KD (Kinase-dead) green-labelled fibroblasts at time zero and 46 hours (corresponding to the wound closure of co-cultured FAK-WT fibroblasts with tumour cells).


migration ( has been analyzed in detail by tracking location of ( n > 16 per condition ) over time for fibroblasts or tumour ( B ) ,

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Cell migration (has been analyzed in detail by tracking the location of cells (n >16 cells per condition) over time for fibroblasts or tumour cells (B),


measuring fibroblast velocity ( C ) , directionality ( a with a directionality equal to 1 is moving straightforward ) ( D ) migration ( E ) or tumour velocity ( F ) , directionality ( G ) ( H ) . Values are means ± of 16 tracked from two independent experiments .

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measuring fibroblast velocity (C), directionality (a cell with a directionality equal to 1 is moving straightforward) (D) and migration distance (E) or tumour cell velocity (F), directionality (G) and distance (H). Values are means ± SEM of 16 tracked cells from two independent experiments.