EMBO/SourceData huggingface dataset:
Figure 5Hs578t cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. SF295 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. NCI H1975 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
Figure 6Twenty-four hours after the last treatment, tumors were excised, frozen and sectioned. Each tumor was stained with Abs specific for either 4HNE, prominin2 or HSF1. Images were taken at 20x magnification. Higher‑magnification images (1.5x) are shown to the right of its corresponding inset (white box). Scale bar is 50 microns. Shown is one representative image for each condition.
Figure 1(A) Picrosirius red (PSR) staining of collagens (red) in PDAC tissue following surgical resection. Scale bar represents 200 µm. (B) Quantification of PSR in low-grade (1-2) and high-grade (3) tumors indicated in panel A as percentage of area. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.(C) Quantification of tumor cell percentage as scored by pathologists in low- and high-grade PDAC indicated in panel A. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.(D) PSR and α-SMA staining of indicated PDAC cell lines which were injected in NSG mice. Scale bar represents 200 µm for PSR and 100 µm for α-SMA images. (E) Quantification of stainings indicated in panel D. Three tumor ROIs of a tumor derived from epithelial cell lines (Capan-2 and AsPC-1) and mesenchymal cell lines (MIA PaCa-2 and PANC-1) are displayed and averages were pooled for analysis. Data information: data are represented as mean±SD, Student's t-test.(F) IHC staining of α-SMA in 3D organotypic co-cultures of PS-1 cells and indicated PDAC cell lines. Scale bar represents 100 µm.
Figure 2(A) Brightfield images of PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) cell lines. Scale bar represents 100 µm.(B) Quantification of bead-calibrated cell count of PS-1 cells after treatment using flowcytometry. n=6 per group, 3 biological replicates for each cell line.(C) EdU assay performed after treatment showing percentage of PS-1 cells in G0/G1 phase, G2/M phase and S phase. n=6 per group, 3 biological replicates for each cell line..
Figure 3(A) Gene expression level of stromal activation markers COL1A1, ACTA2, SPARC, FN1 and FAP in PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) PDAC cell lines using qPCR. Values were normalized to control and n=6 per group, 3 biological replicates for each cell line. Data information: data are represented as mean±SD, Student's t-test.(B) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS-1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m. Values were Log2 transformed and normalized to Suit-2 expression. Data information: data are represented as mean±SD, Student's t-test.(C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm.(D) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.(E) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control (CM of primary CAFs) or mesenchymal PDAC cell lines. Scale bar represents 100 µm.(F) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.
Figure 4(A) Brightfield images of PS-1 cells after 72 hrs of treatment with unfiltered, <100 kDa or <30 kDa filtered CM of indicated cell lines. Scale bar represents 100 µm.(B) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR. Data was normalized against control CM. Statistical significance was tested against unfiltered CM samples. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.(C) Graphical representation of 64 cyto- and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward-phase protein array. Significantly differential expression of proteins (triangles) and genes (green triangles) are identified between epithelial and mesenchymal cell lines. Dotted line indicates P=0.05. Data information: data are represented as mean±SD, Student's t-test.(D) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or epithelial PDAC cells supplemented with recombinant human CSF-1. Scale bar represents 100 µm.(E) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. n=3 biological replicates per group. Data information: data are represented as mean±SD, Student's t-test.(F) PS-1 cells were starved and stimulated with 500 ng/ml rh-CSF-1 at indicated time points. Activation of pathways involved in CSF1R signaling was analyzed using western blot. Samples were derived from the same experiment and blots were processed in parallel.
Figure 5(A) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or mesenchymal PDAC cell lines supplemented with anti-CSF-1. Scale bar represents 100 µm.(B) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.(C) Brightfield images of PS-1 cells after 72 hrs of treatment with PANC-1 CM of pLKOctrl or shCSF1 (#560-562). Scale bar represents 100 µm.(D) Correlation graph of CSF1 expression in PANC-1 pLKOctrl or shCSF1 (#560-562) and how these various CM affect COL1A1 expression in PS-1 cells using qPCR, values were normalized for RPLPO expression. n=3 biological replicates for all individual experiments, error bars indicated SD. R2 and P-value were analyzed with linear regression.(E) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of Capan-2 cells with control vector or Capan-2 cells overexpressing CSF1. Scale bar represents 100 µm.(F) Gene expression level of CSF1 in Capan-2 cells after transduction with control vector or overexpressing CSF1. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.(G) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.
Figure 6(A) IHC staining of CSF-1 in PDAC tissue following surgical resection representative image of CSF-1 expression in grade 1 and grade 3 PDAC is depicted. Scale bar represents 100 µm. (B) For each tumor of panel A, CSF-1 expression in ductal adenocarcinoma cells was quantified. Student's t-test. Shown is mean±SD. n=15 primary PDAC samples.(C) A validation cohort of PDAC patients (n=21) that underwent direct resection was stained for CSF-1 (scale bar represents 100 µm), PSR (scale bar represents 500 µm) and α-SMA (scale bar represents 200 µm). OK8 is low in CSF-1 expression and high in PSR and α-SMA expression, while the opposite accounts for OK7. (D) Correlation of % of PSR in the tumor and CSF-1 expressing tumor cells, as indicated in panel C. R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples (E) Correlation of % of α-SMA in the tumor and CSF-1 expressing tumor cells, as indicated in panel C, R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples
Figure 1D. Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non-precipitated flow-through fractions (FT). IgG shows the presence of anti-PrimPol antibody in the IP fractions (lanes 4-6).
Figure 2B. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-laser irradiated (10 µM TMP, 2 h followed by UVA laser irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.C. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated cells following control or RPA2 downregulation. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.D. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated PRIMPOL KO cells stably expressing V5-tagged WT or RPA binding domain mutant (RBDm) PrimPol versions. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of two assays (n ≥100 cells per condition in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.E. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated WT and BLM KO cells upon downregulation of TOP3A, RMI1 and RMI2 (siTR) when indicated. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥250 cells per condition). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test.
Figure 3H. Immunoblots showing the levels of BLM, FANCD2 and PrimPol after BLM or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in (G). Ponceau-S is shown as loading control.
Figure 4C. Representative confocal microscopy images of EdU staining in WT and PRIMPOL KO cells, 8 h after UVA (control) or TMP-UVA treatment (2 µM TMP, 2 h followed by 5 s irradiation). Nuclear DNA is counterstained with DAPI. When indicated, V5-PrimPol was reintroduced into KO cells. Scale bar, 25 µm. Dot plots indicate the distribution of nuclear EdU intensity, and the mean values are indicated by horizontal red lines. Data from the combination of three replicates (n ≥200 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.
Figure 5A. Confocal microscopy images of FANCD2 immunofluorescence staining in control (UVA) or TMP-UVA-treated (2 µM TMP, 2 h followed by 5 s irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.D. Histograms show the number of chromosomal aberrations per metaphase (average and SEM) in each condition. Data taken from three replicates are combined (50 metaphases per replicate). Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.
Figure 6A. Kaplan-Meier survival curves of WT and PRIMPOL KO mice after the administration of a single dose of MMC (7.5, 10 or 15 mg/kg). Dashed vertical lines represent median survival values for WT (blue) and KO (orange) mice. Statistical analysis was conducted with Log-rank (Mantel-Cox) test; p-value for the highest dose of MMC (15 mg/kg) is indicated.B. Haematoxylin-eosin (H&E) and Ki-67 immunohistochemistry (IHC) stainings of bone marrow sections derived from WT and PRIMPOL KO mice following the administration of 10 mg/kg MMC. Control mice (PBS) were sacrificed when MMC-injected mice reached the humane endpoint. Scale bar, 2.5 mm. In each image, a relevant area is shown at higher magnification (10x).
Figure 7E. IF detection of FANCD2 foci in WT and PRIMPOL KO cells 48 h after TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation) with or without MCM3 downregulation. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.G. Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC or TMP (followed by 90 sec UVA irradiation) for 72 h. The same control curves without MCM3 downregulation are also shown as part of Figure EV6D. Each point in the curve represents the percentage of surviving cells (average and SD of three assays for MMC and two assays for TMP). Statistical analysis was conducted with two-way ANOVA followed by Bonferroni post-test. p-values of comparisons between KO and KO-siMCM3 are indicated.
Figure 1A) Representative zooms of WT MEFs transfected with GFP-tagged Miro1 and Miro2 (GFPMiro1 and GFPMiro2). Control is GFP fused to the first 70 amino acids of Tom70. Tom20 and catalase stain mitochondria and peroxisomes, respectively. Merge is of Miro (green) and catalase (magenta) with colocalisation shown as white. White arrowheads highlight an area where an isolated peroxisome is situated. B) Quantification of the extent of GFP signal on peroxisomes. Data information: B) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.C) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full-length Miro1, but not Miro1 lacking the transmembrane domain.E) Representative images of Miro1 truncation constructs expressed in DKO MEFs. Mitochondria and peroxisomes are stained with Tom20 and catalase, respectively. White arrowheads highlight an area where an isolated peroxisome is situated.F.F) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs. Data information: F) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.G) Co-immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells. GFP-tagged Miro1 truncation constructs were pulled down with GFP-Trap agarose beads and Pex19 was probed with myc antibody.
Figure 2A) Six consecutive frames of live trafficking of peroxisomes (pxDsRed signal at 1.5 seconds per frame). Yellow arrows show the trajectory of a fast-moving peroxisome in both WT and DKO MEFs. Scale bar is 5 μm.B) Blind scoring of the number of long-range peroxisomal trafficking events in WT, Miro1KO and Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For B) a Kruskall-Wallis with a Dunn's correction posthoc test was used to test for significance. For B) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.C) and D) Quantification of individual track length and velocity, respectively (n=24 cells over three independent experiments). Data information: Analysis for C), D) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For C), D) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.E) Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild-type and Miro1-floxed ERT Cre-recombinase MEFs treated with and without 4-OH tamoxifen for 48 hours. Lysates were taken one day after the end of treatment.F) Quantification of long-range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two-minute movie (n=18 cells per condition over three independent experiments). Data information: Statistical significance in F) was calculated by two-way ANOVA. For F) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.G) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.H) Quantification of long-range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a five-minute movie (n=18 cells over three independent experiments). Data information: For H) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.
Figure 3A) Representative images of WT, Miro1KO, Miro2KO and DKO MEFs seeded onto Y-shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (Catalase in white). Scale bar represents 10 μm. Data information: n = 60 cells over three independent experiments.B) Normalised cumulative distribution of mitochondria in WT, Miro1KO, Miro2KO and DKO MEFs. C) Distance at which 95% of mitochondria are distributed (Mito95) for all four genotypes of MEF. Data information: n = 60 cells over three independent experiments. For C) one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.D) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1KO, Miro2KO and DKO MEFs. E) Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox95). Data information: n = 60 cells over three independent experiments. For E) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.
Figure 4A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance.C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments.F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.
Figure 5A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs.B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in B) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments. Data information: Calculation of statistical significance in C) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4).F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments). Data information: Statistical test used in G) was a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.