EMBO/SourceData huggingface dataset:
Figure 3(B) Mean Acidic:Neutral mtKeima per-cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO (Antimycin A (5 µM) and Oligomycin (10 µM)) or three hours with 25 nM BafilomycinA (BafA). Error bars depict S.D. from biological triplicate measurements from three independent clones. Two-way ANOVA with multiple comparisons; p(****)<0.0001.(C) Bar graph showing the number of mtDNA puncta/cell with or without treatment with AO (6 h) in HeLa control or FBXO7-/- cells. Error bars depict S.D. from triplicate technical replicates from two independent clones. One-way ANOVA with multiple comparisons; p(****)<0.0001(D) Indicated hESCs expressing mtKeima were differentiated to iNeurons (day 12) and were subjected to image analysis. Scale bar, 5 μm. (E) The number of red-shifted Keima foci per cell is plotted, where each dot represents the average foci-number per image stack, originating from 7-12 image stacks of iNeuron differentiations. Error bars represent S.D.. One-way ANOVA with multiple comparisons; p(***)=0.0001, p(****)<0.0001.(F) mtKeima mitophagy flux readout of indicated iNeuron genotypes. Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry (>10,000 cells). Pooled data from biological replicates is shown, normalized to 6h BafA treated cells.
Figure 4(A) Workflow for analysis of total protein abundance in HeLa cells expressing Parkin with and without depolarization with AO (16 h). Cell extracts from triplicates were digested with trypsin prior to 18-plex TMT labelling and analysis by mass spectrometry.(F) PINK1 levels in control cells and in two FBXO7-/- clones were measured by TMT-proteomics in fed cells, from biological triplicates from two independent clones.
Figure 5(B) Log2 FC for the indicated mitochondrial protein-groups in control or FBXO7-/- cells at day 0, 4 or 12 during neurogenesis is shown in violin plots. (C) Log2 FC for the indicated cellular organelle proteins in control or FBXO7-/- cells at day 0, 4 or 12 during neurogenesis is shown in violin plots.(F,G) Mitochondrial morphology in iNeurons (iN) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei, in either fed cells or cells treated with AO (3 h) (left panel). The number of median # of mitochondria/cell, median mitochondrial length, and median mitochondrial circularity is shown. Error bars depict S.D. from triplicates from three differentiations (9-12 image stacks each), as shown in panel I left, center, and right, respectively. Two-way ANOVA with multiple comparisons; p(*)=0.0416, p(****)<0.0001. Scale bar = 10 µm.(H) Western Blot analysis on HeLa control and FBXO7-/- whole cell lysate, probed for the mitochondrial fission adapter MiD51.
Figure 3D: KD-values of wild-type, mutant and phosphorylated peptides binding to clathrin NTD (HURP832-846), RANBP2 RanBD4 (DHX38260-275) and SNX27 PDZ (CDC25C30-44). Significant differences indicated by the Mann-Whitney Test (**: p≤0.01). Measurements are from two independent protein preparations with two or three technical replicates each.E: Displacement curves of the KPNA4 ARM domain with the RanBP3114-129 peptides (wild-type, phosphomimetic and phosphorylated) sampling the effects of two distinct phosphosites (T124 and S125). IC50-values for the different peptides are indicated in µM and with SEM. Measurements were obtained in technical triplicates.
Figure 4B: Displacement curves of the three different bait domains and the indicated peptides using wild-type, phosphomimetic and phosphorylated peptide. IC50 values of the different peptides are indicated in µM and with SEM. Measurements were in technical triplicates.
Figure 6A: Displacement curves of the FP experiment using clathrin NTD and wild-type, phosphomimetic and phosphorylated HURP peptides. IC50 values are indicated in in µM and with SEM. Measurements were in four to five technical replicates.F: Co-IP of clathrin with Venus-tagged HURPWT and HURP∆MUT constructs in asynchronous and mitotic HeLa cells.G Co-IP of clathrin with Venus-tagged HURPWT, HURP∆MUT and HURPS839A constructs in mitotic HeLa cells.H: Representative images of HURPWT, HURP∆MUT and HURPS839A in the different mitotic stages.I-J: RNAi rescue experiments (n=2), which included the knockdown of endogenous HURP and re-expressing either RNAi resistant HURPWT, HURP∆MUT and HURPS839A. In order to assess mitotic behaviour of the cells, the time required of control, HURP knockdown, HURPWT, HURP∆MUT and HURPS839A HeLa cells to progress through mitosis (NEBD-anaphase) was established using time-lapse microscopy. Every circle represents one analysed cell. The number of cells analysed is indicated as n in I. Mann-Whitney test was performed to assess significant differences in the median time required for mitotic progression (*: p-value≤0.05, ***: p-value: ≤0.001, ****: p-value ≤0.0001; ns: non-significant)
Figure 1Representative immunofluorescence images of SCOTIN-KO cells expressing Str-Ii-SCOTIN-SBP-EGFP with 40 μM D-biotin treatment for the indicated times. B Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.Representative immunofluorescence images of SCOTIN-KO cells expressing Str-Ii-VSVG-SBP-EGFP (green) with 40 μM D-biotin treatment for the indicated times. , Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.(F) The colocalization of EGFP and Sec61 or CD63 was calculated as the Pearson correlation coefficient from the experimental data in Figure 1E. The error bars indicate the SEMs. n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01 and ****P<0.0001.
Figure 2(A) Immunoblot analysis of proteinase K protection assay. HeLa cells were transfected with SCOTIN-Myc (Total) and digitonin-permeabilized. The supernatants (Cytosol) and pellet (PNSs) were separated by centrifugation. The PNSs were incubated with PBS or with proteinase K (+ PK) in the presence or absence of Triton X-100 (+PK +Triton X).(B) Representative high-resolution microscopy images of Rab5 Q79L-EGFP- and SCOTIN-DsRed-expressing HeLa cells. Magnified images show enlarged endosomes. Ⅰ: SCOTIN on the endosomal membrane, Ⅱ: SCOTIN on the endosomal membrane and luminal side. Scale bar: 10 μm. Scale bars in the magnified images: 5 μm. (C) Plot analysis of SCOTIN relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on lines Ⅰ and Ⅱ from Figure 2B. The arrow indicates the boundary of the endosomal membrane.(F) Left: Representative images of Rab5 Q79L-mCherry-expressing SCOTIN-FP11 knock-in cells. Right: Plot analysis of SCOTIN-FP11 relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on the lines. The arrow indicates the boundary of the endosomal membrane. Scale bars : 10 μm. Scale bars in the magnified images: 5 μm.
Figure 3(B) Representative images from a PLA with VAPA and Rab7 in WT and SCOTIN-Dsred-overexpressing HeLa cells. Only one primary antibody (anti-Rab7 antibody alone) served as a negative control. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.(E) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.(F) Immunoblot analysis of WT and SCOTIN-KO HeLa cell lysates for VAPA, Rab7, SCOTIN, and α-tubulin.(G) Representative images from a PLA with VAPA and Rab7 in SCOTIN-, TMPRD-, or PRD-Myc-transfected SCOTIN-KO HeLa cells. The PLA color legends are inverted. All scale bars: 20 μm. (H) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.
Figure 4SCOTIN PRD-mediated tethering of SUVs and GUVs. E, Representative confocal images of SCOTIN PRD-induced membrane tethering between GUVs and SUVs. PRD-SUV aligned to the PRD-GUV membrane (arrowhead, type I in the model) or accumulated at the surface of GUVs (arrow, type Ⅱ in the model).
Figure 5(B) Representative images of VAPA-Rab7 PLA signals taken from Str-Ii-SCOTIN-SBP-EGFP transfected SCOTIN-KO cells after untreated (-Biotin) or biotin treatment (+ Biotin 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.(D) Representative images of Sec61β-Emerald- or Sec61β-PRD-Emerald-transfected SCOTIN-KO cells. Cells were stained with RTN4 (red). Arrowheads indicate the accumulated ER membrane. All scale bars: 20 μm.(G) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.
Figure 6(C) HEK293 cells were transfected with SCOTIN-GST along with empty (pcDNA3.1-Myc), SCOTIN-Myc, or SCOTIN(Δ150-177)-Myc plasmids for 48 h. SCOTIN-GST was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analyzed by immunoblotting.(D) Representative images from a PLA with VAPA and Rab7 in SCOTIN-KO HeLa cells reconstituted with FL SCOTIN, SCOTIN(ΔPRD), or SCOTIN(Δ150-177)-DsRed. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm.(G) Representative images of VAPA-VAPA PLA signals taken from Str-Ii-SCOTIN(Δ150-177)-SBP-EGFP transfected SCOTIN-KO cells after untreated (-Biotin) or biotin-treated (+Bition 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (H) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.
Figure 7(A-D) TEM images of the ER and endosomes of HeLa (A), SCOTIN-KO HeLa (B), SCOTIN-DsRed transfected SCOTIN-KO HeLa (C), and SCOTIN Δ150-177-DsRed transfected SCOTIN-KO HeLa (D). DsRed-positive cells were processed for EM quantification.
Figure 8(A) Distribution of late endosomes (CD63) in WT or SCOTIN-KO HeLa cells. Representative Z projections of confocal images are shown. The color legends are inverted. Scale bars: 20 μm. (B) Spatial distribution of late endosomes (CD63) expressed as the fractional distance of fluorescent pixels along a straight line drawn through the endolysosomal cloud from the nuclear center (0) to the plasma membrane (1.0) from the experimental data in Figure 8A. (C) Quantification of the number of CD63-positive vesicles per cell from the experimental data in Figure 8A. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.(D) Live-image analysis of WT and SCOTIN-KO HeLa cells expressing Rab7-EGFP. Left: confocal images taken at time 0. Right: time-lapse images of Rab7-EGFP in WT and SCOTIN-KO HeLa cells, as shown by temporal color-coded representations. Each confocal image of late endosomes was color coded according to its acquisition time point, with images taken at 12-sec intervals over 5 min. A total of 24 color-coded images were projected to form a single image that demonstrates the dynamics of Rab7-labeled late endosomes. White = static late endosomes. Multicolored = motile late endosomes. Boxed, magnified images highlight selected perinuclear and peripheral regions. Scale bar: 30 μm. Scale bars in the magnified images: 3 μm. (E) Quantification of the fractional distance of Rab7-EGFP-positive late endosomes in WT and SCOTIN-KO HeLa cells from the experimental data in Figure 8D. Error bars indicate the SEMs. (F) Quantification of Rab7-EGFP-positive late endosome vesicles per cell from the experimental data in Figure 8D. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.(G, H) Live-image analysis of Rab7-EGFP in SCOTIN-KO HeLa cells reconstituted with SCOTIN-DsRed (G) or SCOTIN(Δ150-177)-DsRed (H) taken at time 0 or for 5 min (color-coded images). Lower, boxed, magnified images of selected perinuclear and peripheral regions. Scale bar: 20 μm. Scale bars in the magnified images: 2 μm. (I) Quantification of the fractional distance of Rab7-EGFP in SCOTIN-KO cells reconstituted with SCOTIN-DsRed or SCOTIN(Δ150-177)-DsRed from the experimental data in Figure 8G and 8H. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.
Figure 1B, Representative images (left panel) and quantifications (right panel) of NNMT immunostaining in tissue sections from primary tumors (T) and metastases (M) in different breast cancer models, scale bar: 100 µm. n = 5 tissue sections per model. **P < 0.01; n.s., not significant, n.a., not available; Mann-Whitney U-test. All data are means ± SD.C, Kaplan-Meier overall survival analysis of breast cancer patients stratified according to NNMT protein levels (negative or weak, n = 541; intermediate, n = 108; strong, n = 44) in primary (n = 517) and metastatic (n =176) tumor tissue. Estimated 5-year overall survival rates for subjects with strong, intermediate, and negative/weak NNMT levels were 43.0 ± 10.0%, 69.0 ± 6.0% and 73.0 ± 2.0%, respectively. **P < 0.01; Log-rank test.D, Graphic quantification (pie charts) and representative images of immunostaining from 625 primary breast cancer specimens classified according to NNMT protein abundance [negative (neg)/weak, intermediate or strong]. NNMT levels are significantly higher (P = 0, Student's T-test) in ERα negative (neg) than in ERα positive (pos) breast cancers (side table). Scale bar: 100 µm.E, Kaplan-Meier plot depicting metastasis onset after tumor removal in mice injected orthotopically with SUM159PT WT (n = 10), KO (KOd: n = 5; KOs: n = 5) or KO-NNMT (KOd-NNMT: n= 5; KOs-NNMT: n= 5) cells. *P < 0.05; Log-rank test.F, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 75 after cancer cell injection of MDA-MB-231 KO1- RFP or KO1- NNMT cells and post-primary tumor removal. n = 7 to 10 animals per group. *P < 0.05; Mann-Whitney U-test. All data are means ± SD. G, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 34 after injection of MDA-MB-231 KO1- RFP or KO1- NNMT cells into the tail veins of mice. n = 10 animals per group. ***P < 0.001; Mann-Whitney U-test. All data are means ± SD.
Figure 2C, Representative histological images of SUM159 PT-WT, -KOd, and KOs tumors immunostained with an anti-human vimentin antibody. n = 5 tumors, 2 sections each. Scale bar: 100 µm.G, Representative images of human COL1A1 immunostaining (left panel) and bar graph quantification of COL1A1 positive area (right panel) in tissue sections of SUM159PT WT, KOd, and KOs tumors. n = 10 to 15 tumors per model with 2 - 4 tissue sections per tumor. **P < 0.01; Kruskal-Wallis test. All data are means ± SD. Scale bar: 200 µm.
Figure 3A, Bar graph depicting increased ratio of S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) in SUM159PT KOd and KOs compared to WT cells. n = 3 technical replicates. *P < 0.05; **P < 0.01; Mann-Whitney U-test. All data are means ± SD.D, Dot plots depicting the overlap of H3K9me3 ChIP-sequencing and mRNA-sequencing data. In the y axis: differential H3K9me3 enrichment at the gene promoter in NNMT KO compared to WT cells. In the x axis: differential mRNA expression of NNMT KO compared to WT cells. n = 3 experimental replicates. Cut-off: adjusted P < 0.05. Bold dots highlight collagen genes and PRDM5.F, Bar graph representing average PRDM5 mRNA expression in SUM159PT KOs, KOd, and WT cells. n = 3 experimental replicates with 2 technical replicates each. ****P < 0.0001; Mann-Whitney U-test. All data are means ± SD.G, H, Bar graphs representing average collagen gene mRNA expression upon overexpression (OE) of PRDM5 in SUM159PT KOd and KOs cells. n = 3 to 5 experimental replicates with 2 technical replicates each. *P < 0.05, **P < 0.01, n.s., not significant; Kruskal-Wallis test. All data are means ± SD.
Figure 4A, Representative images and bar graph quantification depicting 5-methylcytosine (5-mC) signal by immunofluorescence in SUM159PT WT, KOs, and KOd cells. n = 2 experimental replicates with 5 to 6 technical replicates each. ***P < 0.001; Kruskal-Wallis test. Data are means ± SD. Scale bar, 50 µm.D, Bar graphs depicting 5-mC abundance at promoters of the indicated genes shown as fold enrichment of methylated DNA immunoprecipitate (MedIP) over IgG control. n = 3 experimental replicates with 2 to 3 technical replicates each. *P < 0.05, ** P < 0.01, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.E, Bar graphs representing average PRDM5 and collagen gene mRNA expression upon 5- aza treatment in SUM159PT WT, Kos, and KOd cells. n = 3 experimental replicates with 2 technical replicates each. *P < 0.05, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.
Figure 5A, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 27 after injection of MDA-MB-231 cells into the tail veins of mice bearing short hairpin sh NT (non-targeting), sh1 or sh2 targeting PRDM5. Mice were fed with doxycycline food to sustain shRNA expression during the whole experiment. n = 7 to 8 animals per group. **P < 0.01; One-way ANOVA. All data are means ± SD.B, Box plots depicting bioluminescence quantification (left panel) and representative images (right panel) of metastases at day 42 and day 62 after intravenous injection of MDA-MB-231 KO1_RFP, KO1_NNMT1 and KO1_COL1A1 (COL1A1 over-expression) cells. n = 4 to 8 animals per group. **P < 0.01, ***P < 0.001; Day 42: One-way ANOVA, Day 62: Student t-test. Boxes define the upper and lower quartiles; central band indicate the median; whiskers define max to min values.E, Box plots showing COL1A1 and PRDM5 expression level (H-Score) according to the NNMT status (NNMT negative, n = 17; NNMT positive, n = 47) within the hormone receptor-negative (HR-) tumor samples. *P < 0.05; Student t-test. Boxes define the upper and lower quartiles; central band indicate the median; whiskers define max to min values.
Figure 1Western blot of pre-mRNA 3'-end processing factors in response to UV treatment (40 J/m2) of A549 cells (n=3), followed by16 hours of recovery. GAPDH was used as a loading control.RT-qPCR assay on nuclear RNA for assessing the uncleaved/total ratio of p53 and TATA-binding protein (TBP) pre-mRNAs in A549 cells (n=3) transfected for 48 hours with two different siRNA targeting PCF11 prior to exposure (UV+) or not (UV-) to UV irradiation (40 J/m2). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.
Figure 2RT-qPCR analysis on RNA extracted from nucleoplasm and chromatin fractions. The ratio of uncleaved pre-mRNA (nucleoplasm/chromatin) was calculated to quantify the level of unprocessed p53, TBP, WDR13 and GAPDH pre-mRNA (n=3 biological replicates) released in the nucleoplasm compared to the chromatin-bound unprocessed pre-mRNA. WDR13 and GAPDH were included as controls as they have been previously reported to be processed post- and co-transcriptionally, respectively. Data are presented as the mean ± s.e.m.RT-PCR analysis of p53 3' flanking region using the same primers employed in the data panel above (n=3). Lane 1 is a control PCR amplification of cDNA derived from the reverse transcription of a control mRNA using oligo (dT). Lanes 2-7 are PCR amplification of reverse transcribed p53 chromatin-associated pre-mRNA using oligo oligo (dT).
Figure 3Representative images of smFISH in untreated (-UV) or UV-treated (+UV) A549 cells with the indicated probes (A, B, C and D).
Figure 4RT-qPCR assay on nuclear RNA for assessing the uncleaved/total ratio of p53 pre-mRNA in wild type (WT) and CoTC deleted (ΔCoTC) A549 cells (n=3) treated with or without UV irradiation (40 J/m2). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.RT-qPCR measuring relative p53 and p21 mRNA levels in wild type (WT) and CoTC-deleted (ΔCoTC) cells (n=3) in response to UV treatment (40 J/m2). The expression was normalized to HPRT. "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.Western blot analysis of p53 and p21 expression in wild type (WT) and CoTC deleted (ΔCoTC) A549 cells (n=3) treated with or without UV irradiation (40 J/m2).
Figure 6RT-qPCR (uncleaved/total RNA) on nuclear RNA extracted from UV-treated or untreated A549 cells (n=3), to assess the regulation of 3'end processing of 20 pre-mRNAs randomly selected from the previous RNA-sequencing data. "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m.RT-qPCR (Released uncleaved pre-mRNA nucleoplasm/chromatin) on RNA extracted from the nucleoplasm and the chromatin fractions of A549 UV-treated cells (n=3). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m.