EMBO/SourceData huggingface dataset:
Figure 7A Representative images of PI3P and PI(3,4)P2 labeling and quantification using mCherry-FYVEHRS and GFP-PH(TAPP1) probes respectively in sh-Control or sh-PI3KC2β hCMEC/D3. Scale bar, 10 µm. Nuclei are shown in blue (DAPI). Staining intensity was quantified by ImageJ software and normalized to total number of cells (DAPI labelling) per field. Data represent mean ± SEM of cell fields randomly selected for PI3P (dots n=21) and for PI(3,4)P2 (dots n=14-16). Data were collected from at least 5 independent experiments; ***p<0.001, unpaired t-test with Welch's correction.C Graph quantification of APPL1-, EEA1- and Rab11- endosomes colocalized with mCitrin-VE-cadherin in sh-Control and sh-PI3KC2β hCMEC/D3 cells. Data are shown as percentage of total count endosomes per cell. Data are expressed as mean ± SEM (n=33-35 cells for APPL1 and EEA1 endosomes and n=16-18 cells for Rab11 endosomes; 3 independent experiments); **p<0.001, Mann-Whitney test or unpaired t-test, as accordance.D Representative confocal microscopic images of VE-cadherin immunostaining in sh-Control cells, sh-PI3KC2β cells or sh-PI3KC2β cells transduced with Rab11S25N (Rab11-DN). Graph quantification of VE-cadherin; control normalized to 100%. Scale bar, 10 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=6-9). Data are collected from at least 3 independent experiments; **p<0.01 Mann-Whitney test.
Figure 1A, MC38 cells in tissue culture were treated with mock (0Gy) and 10Gy irradiation. Conditioned medium (CM) was collected after 48 hours and assayed for the indicated cytokines. Fold-changes in the amounts of the cytokines are shown.B, CSF-1 mRNA expression was measured in MC38 and KPC cells exposed to 10Gy IR compared with mock irradiated cells. Cells were harvested at 24, 48 and 72 hours after irradiation and RNA expression was analysed by RT-qPCR. Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3).C, CSF-1 protein (pg/mg total protein) in CM collected from MC38 and KPC cells 48 hours after exposure to 10Gy IR compared to mock-irradiated cells. Data are presented as mean ± SEM and analysed by Mann-Witney (n=3).D, CSF-1 protein (pg/mg total protein) measured by ELISA in the sera of naïve mice, mice bearing mock-irradiated tumours, and mice bearing irradiated tumours analysed at time points as indicated (n=4/group). Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison.E-F, MC38 (E)and KPC (F) tumours were irradiated with 10Gy. Average tumour volume (red line) is shown with mean TAM infiltrate (blue line) for each time point. For TAM quantification, tumours were disaggregated and CD11b+/F480+ TAMs identified by flow cytometry. Data are presented as mean ± SEM for TAMs and SEM for tumour volume (n=6).G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections; blue=DAPI, green = CD68 (TAMs).I-J, Flow cytometric analysis of immune cell populations within MC38 (I) and KPC (J) tumours 5 days following 10Gy IR compared to mock-irradiated tumours. Macrophages (CD11b+ F480+), neutrophils (CD11b+ Ly6G+), myeloid-derived suppressor cells (CD11b+ Gr1+), CD8 T cells (CD45+ CD3+ CD8+), CD4 T cells (CD45+CD3+CD4+, Natural Killer cells (CD45+ NK1.1+) were identified. Pie charts represent the proportion of CD45+ leukocytes out of the total cells.
Figure 2A, Quantification of iNOS expression on gated macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).B, Quantification of CD206 expression on macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).C, Quantification of the percentage of TAMs (CD11b+ F480+) in MC38 control tumours compared with irradiated tumours (n=6 mice/group).D-E Quantification of iNOShi (D) macrophages as a percentage of total cells in MC38 tumours (n=6).D-E, Quantification of CD206hi (E) macrophages as a percentage of total cells in MC38 tumours (n=6).F, The total number of iNOShi TAMs were divided by CD206hi TAMs to derive a M1/M2 ratio in MC38 and KPC tumours receiving mock or 10Gy irradiation. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).G-H, TAMs (CD11b+F480+) were isolated by FACS. Expression of selected immune stimulatory and immunosuppressive genes in TAMs was determined by RT-qPCR (n=3). Data are presented as mean fold change ± SEM compared to TAMs from mock-irradiated tumours (n=3). Statistical significance was determined by Mann-Witney.I-J, Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation. TAMs were isolated by magnetic bead separation using F480 microbeads and co-cultured at a 1:1 ratio with CFSE labelled CD8+ T cells. CFSE dilution was analysed by flow cytometry to measure proliferation. Percentages of CFSElo T cells were analysed by Kruskal-Wallis with Dunn's multiple comparisons test. Representative histograms are shown (right panel). Experiments were repeated twice for each tumour cell line (n=3 mice/group) *P <0.05 **P <0.01 ***P <0.001.
Figure 3B-C Flow cytometric analysis of TAMs (CD11b+ F480+) in MC38 (B) and KPC (C) tumours receiving indicated treatments. Tumours were harvested 5 days following IR and disaggregated for analysis by flow cytometry. The left panels in B and C show the data derived from the flow cytometry with representative plots shown in the right panels. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group, 3 independent experiments).D-E, Tumour growth kinetics of MC38 (D) and KPC (E) tumours receiving the indicated treatments. Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment.F-G, shows flow cytometric analysis of CD206hi (F) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).F-G, shows flow cytometric analysis of iNOShi (G) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).H-I, CD11b+ F480+ TAMs were isolated from MC38 (H) and KPC (I) tumours 5 days following irradiation (+/-aCSF) and expression of selected immune stimulatory and immunosuppressive genes was analysed by RT-qPCR (n=3). Data are presented as mean ± SEM (10Gy vs 10Gy + aCSF n=6 mice/group). Statistical significance was determined by Mann-Witney. *P <0.05 **P <0.01 ***P <0.001.
Figure 4A-B MC38 (A) and KPC (B) tumours were harvested 5 days following 10Gy IR ± aCSF as indicated. The left panels show the percentage of CD45+ CD3+ CD8+ T cells in these tumours after the indicated treatments. Representative flow cytometry plots from the irradiated groups are shown in the right panels. Tumours that did not receive irradiation were harvested when tumours reached 500mm3. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (A) and Kruskal-Wallis test with Dunn's multiple comparison (B) (n=6 mice/group, 3 independent experiments).C, Flow cytometric analysis of Ki67 expression in the CD8+ T cells in MC38 tumours from A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group).D, Flow cytometric analysis of PD-1 expression on CD8+ T cells from MC38 in tumours as in A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group).E-F Flow cytometric analysis of IFN-γ expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments).E-F, Flow cytometric analysis of Granzyme B expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments).G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections, blue=DAPI, green = CD8.I-J, Tumour growth in CD8 depleted C57BL6 wild-type mice bearing MC38 (I) and KPC (J) tumours receiving treatment as indicated (n=6 mice/group).K, Flow cytometric quantification of intra-tumoural CD8 T cells following anti-CD8 treatment. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group).L-M, Tumour growth in athymic nude mice bearing MC38 (I) and KPC (J) tumours receiving treatments as indicated (n=6 mice/group). Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment. *P <0.05 **P <0.01 ***P <0.001.
Figure 5A-B, CD8+ T cells were isolated from the spleens of MC38 tumour bearing mice. The tumours were radiated with 10Gy and cells were harvested 5 days later. Quantification (A) and representative images (B) of MC38 tumour cell specific tumour-derived CD8 T cell responses detected by IFN-γ ELISpot. The tumour specific CD8+ T cell response was evaluated after T cell incubation with naïve or irradiated MC38 cells for 24 hours. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3 mice/group).C-D, Flow cytometric detection of major histocompatibility complex I (MHCI) expressed on MC38 (C) and KPC (D) tumour cells 48 hours following exposure to 10Gy IR. The left graph shows the overall data, with representative flow cytometry plots on the right. Data presented as mean ± SEM and analysed by Mann-Witney (n=3/group).E-F, Flow cytometric quantification of major histocompatibility complex I (MHCI) expression in vivo. Gated MC38 (E) and KPC (F) tumour cells were analysed 48 hours following exposure to 10Gy IR. Data presented as mean ± SEM and analysed by unpaired t-test (n=3/group).G-H, Flow cytometric quantification of dendritic cells (CD11b+ CD11c+ MHCII+) in MC38 (G) and KPC (H) tumours receiving treatment as indicated and as in E-F. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group).J-K, Tumour growth in mice bearing two MC38 tumours receiving 10Gy IR to the primary lesion (J) ± systemic aCSF therapy (K). The differences in tumour volume 9 days following IR was analysed by unpaired t-test (n=5 mice/group). L-M, Tumour growth in mice bearing two KPC tumours receiving 10Gy IR to the primary lesion (J) ± systemic aCSF therapy (M). The difference in mean tumour volume 10 days following IR was analysed by unpaired t-test (n=8 mice/group).N-O, Flow cytometric analysis of macrophages (N) and CD8 T cells (O) in primary and secondary MC38 tumours. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=5 mice/group). *P <0.05 **P <0.01 ***P <0.001.
Figure 6PD-L1 expression on MC38 and KPC cells 48 hours following 10Gy irradiation in tissue culture (A,B) or 10 Gy irradiation of tumours (C,D) analysed by flow cytometry. Data presented as mean SEM and analysed by Mann-Witney (n=3, A,B). Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group, C,D).E-F, Flow cytometric analysis of PD-L1 (E) and PD-L2 (F) on TAMs in MC38 tumours receiving treatment as indicated above. Data presented as mean SEM and analysed by Mann-Witney (n=5 mice/group)G-H, Tumour growth in mice bearing MC38 (G) and KPC (H) tumours receiving the indicated treatments. Data presented for individual mice. Pie charts indicate the number of regressions observed.I-J, Tumour growth in mice bearing KPC tumours mice receiving 10Gy IR + systemic aPD-L1 to the primary lesion (I) ± systemic aPD-L1 + aCSF therapy (J). The difference in tumour volume 8 (I) or 10 (J) days following IR was analysed by unpaired t-test (n=8 mice/group). *P <0.05 **P <0.01 ***P <0.001.
Figure 1A. Characteristics of IGR39 /IGR37 and WM115/WM266.4 melanoma cell lines (Upper Panel). Pigmentation of melanoma cells pellets (lower panel).B. Growth curve of primitive IGR39 and metastatic IGR37 cell line (N=3 biological replicates). Data are mean ±SD.C. Phase-contrast images of IGR39 and IGR37 cells. Scale bar is 100µm.D. Analysis of IGR39 and IGR37 speed of migration by time-lapse microscopy.
Figure 2B. Scatter plot of identified and quantified proteins in the secretome of primitive IGR39 and metastatic IGR37. Red dots represent proteins that were significant with FDR <0.05, blue dots represent proteins with p-value<0.05. C. Scatter plot of identified and quantified proteins fin the secretome of primitive WM115 and metastatic WM266.4. Red dots represent proteins that were significant with FDR <0.05, blue dots represent proteins with p-value<0.05. D. (Left)Venn diagram of the significant proteins shared by both IGR and WM cell lines. (Right) Histograms representing the metastatic and primitive signatures H/M ratios.F. Confocal fluorescence images of Proteostat (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. G. Quantitation of aggregates/cell in IGRs cell lines by immunofluorescence analysis, left panel; fluorescence gain of soluble proteins treated with Proteostat reagent, right panel. (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).
Figure 3A. Absolute quantitation of 24, 25 and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***p-value<0.001, N=3 biological replicates, data are mean ±SD).B. Histogram representing R square of correlation analysis between absolute quantitation of 24, 25 and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines.C. Confocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10µm.D. Western blot on IGR37 and 39 cellular lysates probed with anti-HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates.
Figure 4A-D. Immunofluorescence images with Proteostat (red) and DAPI (blue) staining on (A) human normal skin, (B) samples of primitive melanomas, (C) melanoma metastases in brain and (D) melanoma metastases in lung.E. Details of brain metastases. Scale Bar is 30µm. F. Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues: 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed. For each tissue two sections were quantified. T-test analysis was applied. T-test analysis, ** = p-value<0.01 (N=6, data are mean ±SEM).
Figure 5A. Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB-360.B. Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB-360.D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD). F. Quantitation, by immunofluorescence analysis, of YAP in different cellular compartments (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). Images were quantified by subdividing cells into mostly cytosolic YAP (Cytosol), mostly nuclear YAP (Nuclear), or equal distribution (Total) from three biological replicates.G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).H. CTGF mRNA level measured by real-time PCR in IGR39 treated with IGR39 conditioned medium (CTRL) or with IGR37 conditioned medium (CM), N=4 biological replicates. T-test analysis, *** p-value <0.001, data are mean ±SD.I. CTGF mRNA level measured by real-time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis: *** p-value <0.001, data are mean ±SD.J. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.
Figure 6A. MTT assay for IGRs treated with DMSO or NB-360 (25µM). Biological replicates N=4 biological replicates. T-test analysis ** = p-value<0.01, *** p-value <0.001. Data are mean ±SD. B. MTT assay for IGRs cells treated with DMSO or inhibitor 3I (3µM). Biological replicates N=3 biological replicates. T-test analysis ** = p-value<0.01. Data are mean ±SD. C-D. MTT assay of IGRs treated with NB-360 (25µM) and different concentration of doxorubicin as indicated. Biological replicates N=4 biological replicates. T-test analysis: * = 0.01<p-value<0.05, ** = p-value<0.01. Data are mean ±SD.E. T-test analysis of IC50 values for doxorubicin used alone or in combination with NB-360 (combo). Data are mean ±SD. T-test analysis: * = 0.01<p-value<0.05; ** = 0.001<p-value<0.01.
Figure 1p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown.Induction of selected p53 target genes after DNA damage induction in A549 wild-type and p53 knockdown cells. RNA levels were measured by qRT-PCR before and 3h after treatment with 10 Gy R. Fold changes relative to basal levels are shown for each cell line.Fluorescence microscopy images of smFISH probes labelled with CAL-Fluor 610 (grey) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets shows broad variability of RNA counts per cell for all genes in basal conditions.
Figure 2Fluorescence microscopy images of smFISH probes CAL-Fluor 610 (grey) overlayed with Hoechst 33342 staining (blue) at 3 h after 10 Gy IR or the indicated target genes in A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.We quantified RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). smFISH based single cell analysis of gene expression patterns highlights distinct RNA counts for p53 targets. RNA counts per cell are displayed as boxplots (see Data Visualization section); n: number of analyzed cells, fc: median fold of induction relative to time point basal (indicated by grey lines) Distributions of RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Despite a clear change in median levels (m: median), single cell analysis reveals a strong dispersion that overlaps for the different conditions, as shown by the strongly overlapping distributions of RNA counts per cells. For better visualization, probability density estimates (PDF) based on a normal kernel are shown
Figure 3Quantification of stochastic gene expression for the indicated p53 target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (µ) (proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity (> 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates µ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2C).Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene.Amount of p53 bound to indicated target gene promoters before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time-point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT-PCR measurements) from 3-4 biological repeats are shown as dots, mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.)
Figure 4Chk2 inhibition with the small molecule BML-277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML-277 by immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).We quantified promoter activity of BAX (B, sustained archetype) and PPM1D (C, pulsatile archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The fraction of active promoters was reduced at 6 h and 9 h after irradiation; the transcription rate was not notably affected.Sequential treatment with Nutlin-3 converts pulsatile p53 dynamics into sustained nuclear levels. A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0.75 µM Nutlin-3 at 2.5 h, with 2.25 µM at 3.5 h and 4 µM at 5.5 h post IR based on immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).We quantified promoter activity of MDM2 (E, transient archetype) and CDKN1A (F, transient archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and sequential Nutlin-3 treatment. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The relative fraction of active promoters strongly increased, changing transient to sustained archetypes. The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics (inset, fold change relative to IR alone for each time point).