C . Overexpression of chicken RNase H1 prevents ( GAA ) 10 induced BU - 1A epigenetic instability in primpol . Fluctuation was performed on three primpol BU - 1A ( GAA ) 10 clones . One - way ANOVA was used to calculate significance of differences in BU - 1 instability between primpol BU - 1A ΔG4 other lines . * * * * p ≤ 0 . 0001 , ns = not

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C. Overexpression of chicken RNase H1 prevents (GAA)10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA)10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significant


D . Diagram of RNase H1 hybrid binding domain ( HBD ) - mCherry fusion flow cytometry expression profiles of construct in four

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D. Diagram of the RNase H1 hybrid binding domain (HBD) - mCherry fusion and flow cytometry expression profiles of the construct in four clones


E . R - loop stabilisation induces epigenetic instability of BU - 1 . Bu - 1a fluctuation of wild type expressing HBD - mCherry . scatter plots pool results from at least two different clones with matched HBD - expression . Mean ± SD reported . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , ns = not significant ; one - way ANOVA .

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E. R-loop stabilisation induces epigenetic instability of BU-1. Bu-1a fluctuation analysis of wild type cells expressing HBD-mCherry. The scatter plots pool results from at least two different clones with matched HBD-expression. Mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant; one-way ANOVA.


A . Expression of geminin - tagged chicken RNase H1 - YFP . Phases of cycle were determined by content in live by Hoechst 33342 ( X - axis ) . RNase H1 - YFP with or without geminin degron is detected on Y - axis . RNase H1 - YFP - geminin degron is degraded in G1 . In contrast , RNase H1 - YFP remain stable irrespective of phase of cycle . 2n 4n indicate chromosome number before after

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A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication


B . Bu - 1a fluctuation of two independently derived primpol BU - 1A ( GAA ) 10 : Gg RNase H1 - YFP - geminin degron clones . Since expression of RNase H1 - YFP - geminin degron construct is not stable ( unlike RNase H1 - YFP construct without degron ) , Bu - 1a expression was assessed separately in YFP + ve YFP - ve within each clone . Statistical differences calculated Kruskal - Wallis . For panels , at least 36 clones were analysed ; mean ± SD reported . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , ns = not

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B. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA)10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant


C . DRIP - qPCR for R - loops around engineered + 3 . 5 ( GAA ) 10 repeat in BU - 1 in different phases of cycle . location of qPCR amplicons is indicated in map at top of panel . BU - 1 DRIP signal was normalised to - 0 . 5 kb amplicon in G1 - arrested ( t = 0 h ) Black : wild type ; red : primpol . Error bars = SD . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 .

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C. DRIP-qPCR for R-loops around the engineered +3.5 (GAA)10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h) Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05


E . Validation of of nascent : hybrid formation in BU - 1 locus . Enrichment of 4SU labelled moiety of : hybrids was calculated relative to input in three independent asynchronous wild type ( black ) or primpol ( red ) , with or without exogenous RNase H treatment . Error bars = SD . * * p ≤ 0 . 01 , * p ≤ 0 . 05 , ns = not significant ; unpaired t -

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E. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-test


G . Newly synthesised R - loops in BU - 1 during S phase in wild type ( black ) primpol ( red ) . Error bars represent 1SD of three biological repeats of experiment . * * * p ≤ 0 . 001 , * p ≤ 0 . 05 ; unpaired t -

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G. Newly synthesised R-loops in BU-1 during S phase in wild type (black) and primpol (red). Error bars represent 1SD of three biological repeats of the experiment. *** p ≤ 0.001, * p ≤ 0.05; unpaired t-test


A . Representative normalised DRIP - seq data in two genes COL22A1 , spanning over 200 kb , MYC . locations of H - G4 motifs are shown below gene map . Wild type in blue ; primpol in red . corresponding RNAse H - treated samples are dashed . See Methods for further details of graphic generation . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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A. Representative normalised DRIP-seq data in two genes COL22A1, spanning over 200 kb, and MYC. The locations of H-DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNAse H-treated samples are dashed. See Methods for further details of graphic generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


C . DRIP peak heights in wild type primpol DT40 normalised to Drosophila S2 spike - in . n ( wild type ) = 41445 ; n ( primpol ) = 48648 . D . Correlation of normalised DRIP peak heights in overlapping peaks between wild type primpol . Blue = 1 : 1 correlation ; red = linear regression through data . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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C. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike-in. n (wild type) = 41445; n (primpol) = 48648. D. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


E . Correlation between H - forming sequences genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . F . Normalised DRIP peak heights in genes identified as associating with H - . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). F. Normalised DRIP peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


G . Correlation between G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . H . Normalised DRIP peak heights in genes identified as associating with G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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G. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). H. Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


A . Representative normalised DIP - seq data in SKI locus . Wild type in blue ; primpol in red . locations of H - G4 motifs are shown below gene map . corresponding RNAse H - treated samples are dashed . Since so little material was recovered following RNase H treatment , samples were pooled prior to library generation . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

A. Representative normalised RNA DIP-seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H-DNA and G4 motifs are shown below the gene map. The corresponding RNAse H-treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


C . DIP - seq peak heights in wild type primpol BOBSC normalised to a DT40 spike - in . n ( wild type ) = 32740 ; n ( primpol ) = 33721 . D . Correlation of normalised DIP - seq peak heights in overlapping peaks between wild type primpol . Blue = 1 : 1 correlation ; red = linear regression through data . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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C. RNA DIP-seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike-in. n (wild type) = 32740; n (primpol) = 33721. D. Correlation of normalised RNA DIP-seq peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


E . Correlation between H - forming sequences genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


F . Correlation between G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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F. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


G . Normalised DIP - seq peak heights in genes identified as associating with H - . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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G. Normalised RNA DIP-seq peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


H . Normalised DIP - seq peak heights in genes identified as associating with G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

H. Normalised RNA DIP-seq peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


A Immunohistochemistry was used to assess STAMP2 expression in normal malignant human prostate specimens . Tissue microarrays ( TMAs ) with normal prostate ( n = 27 ) early - stage ( Gleason grade < 7 ) ( n = 35 ) or late - stage prostatetumors ( Gleason grade ≥ 7 ) ( n = 149 ) were subjected to immunohistochemistry as described in Materials Methods . Representative are shown . B of cytosolicSTAMP2 in TMAs is shown in ( A ) . One - way of variance ( ANOVA ) with a post hoc , * P < 0 . 0001 . Error bars indicate . C , D details of ( C ) ( D ) STAMP2 in TMAs described in ( A ) .

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A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens. Tissue microarrays (TMAs) with normal prostate (n = 27) and early-stage (Gleason grade < 7) (n = 35) or late-stage prostatetumors (Gleason grade ≥ 7) (n = 149) were subjected to immunohistochemistry as described in Materials and Methods. Representative images are shown.B Quantification of cytosolicSTAMP2 staining in TMAs is shown in (A). One-way analysis of variance (ANOVA) with a post hoc test, *P < 0.0001. Error bars indicate SEM.C, D The details of cytosolic (C) and membrane (D) STAMP2 staining in the TMAs described in (A).


A LNCaP were transfected with either control siRNA or two independent STAMP2 siRNAs , fractions of were prepared followed by Western blotting . STAMP1 is used as a loading control .

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A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs, and membrane fractions of the cells were prepared followed by Western blotting analysis. STAMP1 is used as a loading control.


B LNCaP were cultured in RPMI 1640 medium containing 10 % CT - FBS treated with or without 1 nM R1881 for 24 h before being transfected with indicated siRNAs . were then cultured for indicated times , growth was measured by Counting Kit - 8 . n = 3 , * P < 0 . 0001 . Error bars indicate SD .

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B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD.


C LNCaP were cultured transfected with indicated siRNAs as described in ( B ) . After transfection , were cultured for 10 days . colonies formed were stained photographed .

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C LNCaP cells were cultured and transfected with the indicated siRNAs as described in (B). After transfection, the cells were cultured for 10 days. The colonies formed were stained and photographed.


D LNCaP stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881 . were then collected , fractions were prepared followed by Western blot .

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D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis.


E LNCaP stably expressing shRNA against STAMP2 or control shRNA were cultured for indicated times , numbers were determined using a hemocytometer . n = 3 , * P = 0 . 047 ; * * P = 0 . 0046 . Error bars indicate SD .

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E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times, and the cell numbers were determined using a hemocytometer. n = 3, *P = 0.047; **P = 0.0046. Error bars indicate SD.


F LNCaP stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 as described in Materials Methods . plates were then stained photographed . G of data from ( F ) . n = 3 , * P = 0 . 022 . Error bars indicate SD .

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F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD.


H LNCaP stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into SCID mice . was measured after 8 . n = 9 , * P = 0 . 037 . Error bars indicate .

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H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice. Tumor size was measured after 8 weeks. n = 9, *P = 0.037. Error bars indicate SEM.


I VCaP were transfected with indicated siRNA as described in ( B ) . After transfection , VCaP were cultured for 2 . colonies formed were stained photographed . J of data from ( I ) . n = 3 , * P < 0 . 0001 . Error bars indicate SD .

I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.


A LNCaP were transfected with either control or STAMP2 - specific siRNA . Two days after transfection , were subjected to cycle as described in Materials Methods . Representative histograms showing PI - stained are shown . B proportion of in each stage of cycle from experiment in ( A ) is presented . Student ' s t - was performed to analyze statistical significance , n = 3 . * P = 0 . 0002 ; * * P = 0 . 0056 ; * * * P = 0 . 0099 . Error bars indicate SD .

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A LNCaP cells were transfected with either control or STAMP2-specific siRNA. Two days after transfection, cells were subjected to cell cycle analysis as described in Materials and Methods. Representative histograms showing PI-stained cells are shown.B The proportion of cells in each stage of the cell cycle from the experiment in (A) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0002; **P = 0.0056; ***P = 0.0099. Error bars indicate SD.


C Two days after transfection with indicated siRNAs , LNCaP were harvested lysates were prepared subjected to Western blot with indicated antisera . Representative blots of three independent experiments are shown .

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C Two days after transfection with the indicated siRNAs, LNCaP cells were harvested and cell lysates were prepared and subjected to Western blot analysis with the indicated antisera. Representative blots of three independent experiments are shown.


D LNCaP were transfected with either control or STAMP2 - specific siRNA . Three days after transfection , were treated with either 50 ng / ml TRAIL or 20 μmol / l LY294002 ( LY ) for 24 h , or both agents for 6 h , then subjected to TUNEL / FACS . Representative histograms of TUNEL - stained are shown . FL1 - H refers to gating of for TUNEL with measurement . E extent of apoptosis from experiment in ( D ) is presented . Student ' s t - was performed to analyze statistical significance , n = 3 . * P = 0 . 027 ; * * P = 0 . 0058 ; * * * P = 0 . 0016 . Error bars indicate SD .

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

D LNCaP cells were transfected with either control or STAMP2-specific siRNA. Three days after transfection, cells were treated with either 50 ng/ml TRAIL or 20 μmol/l LY294002 (LY) for 24 h, or both agents for 6 h, and then subjected to TUNEL/FACS analysis. Representative histograms of TUNEL-stained cells are shown. FL1-H refers to the gating of the cells for the TUNEL staining with fluorescence measurement.E The extent of apoptosis from the experiment in (D) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.027; **P = 0.0058; ***P = 0.0016. Error bars indicate SD.


F LNCaP were transfected with either control or two independent STAMP2 - specific siRNAs , ST2 - 1 ST2 - 2 . Three days after transfection , were treated in same way as in ( D ) . Whole - lysates were prepared subjected to Western blot with indicated antisera .

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F LNCaP cells were transfected with either control or two independent STAMP2-specific siRNAs, ST2-1 and ST2-2. Three days after transfection, cells were treated in the same way as in (D). Whole-cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.


G LNCaP stably expressing either control or STAMP2 vector were treated in same way as in ( D ) . lysates were prepared subjected to Western blot with indicated antibodies .

G LNCaP cells stably expressing either control or STAMP2 vector were treated in the same way as in (D). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies.


A STAMP2 expression was determined by of a neoadjuvant hormone therapy ( NHT ) TMA containing samples from hormone naïve ( untreated ) ( n = 28 ) , NHT 1 - 6 months ( n = 31 ) , NHT 7 - 12 months ( n = 41 ) , with PSA recurrence ( n = 21 ) as indicated . Representative are presented . B Quantitative presentation of data from ( A ) . Unt , untreated ; Recur , PSA recurrence . Kruskal - Wallis was used for significance . * P = 0 . 0499 . Error bars indicate .

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A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy (NHT) TMA containing samples from hormone naïve (untreated) (n = 28), NHT 1-6 months (n = 31), NHT 7-12 months (n = 41), and patients with PSA recurrence (n = 21) as indicated. Representative images are presented.B Quantitative presentation of the data from (A). Unt, untreated; Recur, PSA recurrence. The Kruskal-Wallis test was used for significance analysis. *P = 0.0499. Error bars indicate SEM.


C Boxplots of STAMP2 expression in normal prostate tissue , prostate cancer , metastatic CRPC were analyzed in two independent GEO datasets ( GSE6919 GSE35988 ) . values of samples are presented as dots . Thick horizontal lines represent median , with box representing upper lower quartile . whiskers represent 5th 95th percentiles . statistical significance , indicated in tables at bottom , was determined by Student ' s t - .

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C Boxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test.


D CWR22 xenografts were grown in nude mice , tumors were collected at different times after castration . mRNA was extracted from tumors used for qPCR of STAMP2 expression . results are presented as boxplots . Thick horizontal lines represent median , with box representing upper lower quartile . whiskers represent 5th 95th percentiles , outlier is presented as an open circle . statistical significance was determined by one - way ANOVA with a post hoc . n = 3 in group week 0 , 1 , 2 , 4 ; n = 5 in refractory group . * P = 0 . 011 ; * * P = 0 . 049 . Error bars indicate .

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D CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression. The results are presented as boxplots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles, and the outlier is presented as an open circle. The statistical significance was determined by one-way ANOVA with a post hoc test. n = 3 in group week 0, 1, 2, and 4; n = 5 in refractory group. *P = 0.011; **P = 0.049. Error bars indicate SEM.


A 22Rv1 were cultured in RPMI 1640 medium containing 10 % CT - FBS treated with or without 1 nM R1881 for 24 h were then transfected with indicated siRNAs . were then cultured for 10 days . colonies formed were stained photographed . B of data from ( A ) . Student ' s t - was performed to analyze statistical significance , n = 3 . * P = 0 . 0003 ; * * P < 0 . 0001 . Error bars indicate SD .

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A 22Rv1 cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h and were then transfected with the indicated siRNAs. The cells were then cultured for 10 days. The colonies formed were stained and photographed.B Quantification of data from (A). Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0003; **P < 0.0001. Error bars indicate SD.


C 22Rv1 from Supplementary Fig S2 were subcutaneously implanted into both flanks of nude mice ( six mice per group ) . was measured at indicated time points . Student ' s t - was performed to analyze statistical significance , n = 10 . * P = 0 . 037 ; * * P = 0 . 039 . Error bars indicate . D Representative tumors from ( C ) right after resection at final time point are shown .

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C 22Rv1 cells from Supplementary Fig S2 were subcutaneously implanted into both flanks of male nude mice (six mice per group). Tumor size was measured at the indicated time points. Student's t-test was performed to analyze the statistical significance, n = 10. *P = 0.037; **P = 0.039. Error bars indicate SEM.D Representative tumors from (C) right after resection at the final time point are shown.


A LNCaP were transfected with either control or STAMP2 - specific siRNA in presence of 10 − 8 M R1881 . was isolated , qPCR was used to determine ATF4mRNA . Student ' s t - was used to analyze statistical significance , n = 3 . * P = 0 . 002 . Error bars indicate SD .

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A LNCaP cells were transfected with either control or STAMP2-specific siRNA in the presence of 10−8 M R1881. RNA was isolated, and qPCR was used to determine ATF4mRNA levels. Student's t-test was used to analyze the statistical significance, n = 3. *P = 0.002. Error bars indicate SD.


B lysates from ( A ) were subjected to Western blot .

B Cell lysates from (A) were subjected to Western blot analysis.


C , D ATF4 expression in LNCaP ( C ) 22Rv1 ( D ) stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot .

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C, D ATF4 expression in LNCaP (C) and 22Rv1 (D) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis.


E ATF4 expression in xenografted tumors of 22Rv1 stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR . Student ' s t - was used to analyze statistical significance , n = 4 . * P = 0 . 022 ; * * P = 0 . 015 ; * * * P = 0 . 044 . Error bars indicate .

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E ATF4 expression in xenografted tumors of 22Rv1 cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR. Student's t-test was used to analyze the statistical significance, n = 4. *P = 0.022; **P = 0.015; ***P = 0.044. Error bars indicate SEM.


F CWR22 xenografts were grown in nude mice , tumors were collected at different times after castration . association between ATF4 STAMP2 in samples was determined by Western blot .

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F CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. The association between ATF4 and STAMP2 in the tumor samples was determined by Western blot analysis.


G MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from cBio Cancer Genomics Portal . was performed as described in Materials Methods . expression of STAMP2 ATF4 , as well as two ATF4 target genes ( ASNS SLC7A11 ) , are presented .

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G The MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from the cBio Cancer Genomics Portal. Analysis was performed as described in Materials and Methods. The expression levels of STAMP2 and ATF4, as well as two ATF4 target genes (ASNS and SLC7A11), are presented.


A LNCaP were transfected with either control or ATF4 siRNA . Two days after transfection , were harvested whole - lysates were made used in Western blot .

A LNCaP cells were transfected with either control or ATF4 siRNA. Two days after transfection, cells were harvested and whole-cell lysates were made and used in Western blot analysis.


B LNCaP were transfected with either control or ATF4 siRNA were cultured for 2 . colonies formed were stained photographed . C covered by colonies on each plate in ( B ) was quantified represented as of total of plate . * P = 0 . 0023 .

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B LNCaP cells were transfected with either control or ATF4 siRNA and were cultured for 2 weeks. The colonies formed were stained and photographed.C The area covered by the colonies on each plate in (B) was quantified and represented as percentage of the total area of the plate. *P = 0.0023.


D lysates of LNCaP stably expressing an empty vector ( Vector ) or a vector expressing ATF4 ( ATF4 ) were prepared subjected to Western blot with indicated antisera .

D Cell lysates of LNCaP cells stably expressing an empty vector (Vector) or a vector expressing ATF4 (ATF4) were prepared and subjected to Western blot analysis with the indicated antisera.


E After transfection with indicated siRNAs , from ( D ) were cultured for 2 . colonies formed were stained photographed . F of colonies in ( E ) was quantitated using an image system represented as of total of plate . * P = 0 . 024 .

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E After transfection with the indicated siRNAs, cells from (D) were cultured for 2 weeks. The colonies formed were stained and photographed.F The area of the colonies in (E) was quantitated using an image analysis system and represented as percentage of the total area of the plate. *P = 0.024.


G LNCaP were transfected with either control ( Ctrl ) or ASNS siRNA . were then cultured for 2 . colonies formed were stained photographed . Knockdown of ASNS was confirmed by Western blot shown at top . H of data shown in ( G ) . * P = 0 . 0087 .

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G LNCaP cells were transfected with either control (Ctrl) or ASNS siRNA. The cells were then cultured for 2 weeks. The colonies formed were stained and photographed. Knockdown of ASNS was confirmed by Western blot analysis shown at the top.H Quantification of the data shown in (G). *P = 0.0087.


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 .

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 .

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 .

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 .

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 ,

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 ) .

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 .

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 .

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 .

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.