K . Basal R - GECO1 fluorescence ( F ) normalized to the maximum R - GECO1 fluorescence ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . L . Average basal R - GECO1 fluorescence ( F0 ) normalized to the maximum R - GECO1 fluorescence intensity ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

K. Basal R-GECO1 fluorescence (F) normalized to the maximum R-GECO1 fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. L. Average basal R-GECO1 fluorescence (F0) normalized to the maximum R-GECO1 fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


B . Flow cytometry for Bu - 1a expression in wild type cells with ( GAA ) n tracts of different length knocked into the BU - 1A locus ( in blue ) . DT40 cells are heterozygous and carry one BU - 1A and one BU - 1B allele . All experiments introducing repeats into BU - 1A are carried out in cells in which the + 3 . 5 G4 has been deleted from both A and B alleles , to avoid transvection between the allele Black outline : positive control ( wild type cells ) ; red outline : negative control ( cells carrying a genetic disruption of BU - 1 )

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

B. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1)


C , D . Bu - 1a fluctuation analysis of wildtype and primpol cells in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) or with ( GAA ) 10 and ( GAA ) 20 sequence orientated such that it is replicated as the leading ( C ) or lagging ( D ) strand template for a fork entering from the 3 ' end of the locus as shown in panel ( A ) . At least two independent fluctuation analyses were performed . Circles represent the percentage of Bu - 1a loss variants in at least 24 individual clones from these experiments , with mean ± SD reported . * * * * p < 0 . 0001 , * p ≤ 0 . 05 , ns = not significant ; one - way ANOVA .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.


Human PrimPol , or mutants , tagged with YFP were expressed in primpol cells harbouring ( GAA ) 10 sequence in the BU - 1A locus . Bu - 1a and YFP double positive cells were sorted and expanded for 2 weeks , and then analysed for Bu - 1a expression variants . For each complementation , at least two independently derived clones were subjected to fluctuation analysis . As previously observe expression of hPrimPol [ AxA ] and hPrimPol [ ZfKO ] is deleterious and unstable . Cells expressing these mutations and remaining YFP - positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis . Pooled results from at least three independent fluctuation analyses are represented with mean ± SD indicated with red bar and whiskers . Statistical significance : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p ≤ 0 . 05 , ns = not significant ; Kruskal - Wallis

O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observe expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test


A . DRIP - qPCR analysis reveals accumulation of R - loops across the BU - 1 locus in primpol cells . The DRIP signal was calculated as enrichment over RNase H - treated samples and was normalised to - 0 . 5 kb amplicon . The mean and SD for three biological replicates is presented . An unpaired t - test was used to compare differences between matched amplicons in primpol BU - 1A ( GAA ) 10 and the other cell lines indicated . * * * * p ≤ 0 . 0001 , ns = not

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

A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant


B . DNA : RNA hybrids in primpol BU - 1A ( GAA ) 10 : GgRNase H and primpol BU - 1A ( GAA ) 10 : hPrimPol . An unpaired t - test on three biological replicates was used to compare differences to primpol BU - 1A ( GAA ) 10 for each matched amplicon . The bar represent the mean and whiskers the SD . * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , ns = not

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

B. DNA:RNA hybrids in primpol BU-1A(GAA)10:GgRNase H and primpol BU-1A(GAA)10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA)10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significant


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

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

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

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

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 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 .

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

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 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

O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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 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

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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 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

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 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 -

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

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 ) and primpol ( red ) . Error bars represent 1SD of three biological repeats of the experiment . * * * p ≤ 0 . 001 , * p ≤ 0 . 05 ; unpaired t -

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

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 , 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 ) .

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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 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 ) .

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

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 - 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 ) .

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

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 ) 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 ) .

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

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 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 ) .

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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 . 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 ) .

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

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 - 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 ) .

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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 ) 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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

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


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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the cells expressing the different constructs used in ( A ) as described in Materials and 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 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 .

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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 the cells from ( C ) as described in Materials and 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 cell line with Dox - inducible STAMP2 expression or vector control was generated . Western blot analysis 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 The cells from ( E ) were either left untreated or treated with increasing amounts of Dox , and ferrireductase activity was determined . * P < 0 . 0001 .

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the cells described in ( A ) . * P = 0 .

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells 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 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 ) .

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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 ) and H3K9me3 ( red ) in the indicated migration conditions . Images 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 all indicated A375 conditions ( Control ( n = 59 ) , Top - 5 ( n = 64 ) , Bottom - 5 ( n = 62 ) nuclei ) . Kolmogorov - Smirnov test 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 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 .

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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 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 .

(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 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 .

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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 ) 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 .

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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 ) 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 ) .

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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 ) and Bottom - 5 ( n = 109 ) cells embedded in 3D collagen matrices . Kolmogorov - Smirnov test 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 ) 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 )

(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 .

The structure of SARS-CoV-2 monomer.


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

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


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 ) .

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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 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 .

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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 images of GINs from CTRL and sMDD at day 65 .

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


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 .

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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 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 ,

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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 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 .

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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 ) 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 ) .

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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 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 .

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 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 ) .

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

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


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 .

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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 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 .

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

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


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 .

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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 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 .

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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.


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 .

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 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 .

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

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.


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 .

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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 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 .

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.


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 .

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 images of calcium imaging in CTRL ( from RC01001 - C ) and sMDD ( from SA004 ) GINs at resting and 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.


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 .

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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 the expression level and percentage of genes selected from functional gene sets across CTRL , sMDD and 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 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 .

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

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

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


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 .

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 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

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.


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 ) .

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 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

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).


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 .

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

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


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 .

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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 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 ) .

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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 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 ) .

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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).


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 .

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

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 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 .

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

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 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 .

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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 % ) 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 .

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

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 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 .

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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 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 .

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

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 ) 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 .

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

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 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 ) .

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 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

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).