Quantification was performed to determine number of positive vesicles per cell ( ImageJ software ) . Nuclei are shown in blue ( DAPI ) . Data represent mean ± SEM of cell fields randomly selected ( n = 13 - 20 for ( C ) and n = 11 - 20 for ( D ) ) . Data are collected from at least 3 - 4 independent experiments . For violin plots ( D ) , thick lines represent median and thin lines interquartile ; * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 , unpaired t - test or Mann - Whitney , as accordance .

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Quantification was performed to determine number of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.


( A ) Representative VE - cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field ( DAPI labelling ) in sh - Control and sh - PI3KC2β hCMEC / D3 in resting condition and after 24 hours activation with 25 ng / ml of TNFα . Control normalized to 100 % . Scale bar , 15 µm . Data represent mean ± SEM of cell fields randomly selected ( dots n = 14 - 19 ) . Data are collected from 4 - 5 independent experiments ; * * * p < 0 . 001 , unpaired t - test .

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(A) Representative VE-cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field (DAPI labelling) in sh-Control and sh-PI3KC2β hCMEC/D3 in resting condition and after 24 hours activation with 25 ng/ml of TNFα. Control normalized to 100%. Scale bar, 15 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=14-19). Data are collected from 4-5 independent experiments; ***p<0.001, unpaired t-test.


( B ) Graph quantification of fluorescence intensity of VE - Cadherin normalized to total cell number per field ( DAPI labelling ) in control condition and after 5 hours in oxygen - glucose deprivation ( OGD ) condition . Control normalized to 100 % . Data represent mean ± SEM of cell fields randomly selected ( dots n = 16 ) . Data were collected from 5 independent experiments ) ; * * * p < 0 . 001 ; * * p < 0 . 01 , Mann - Whitney test .

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(B) Graph quantification of fluorescence intensity of VE-Cadherin normalized to total cell number per field (DAPI labelling) in control condition and after 5 hours in oxygen-glucose deprivation (OGD) condition. Control normalized to 100%. Data represent mean ± SEM of cell fields randomly selected (dots n=16). Data were collected from 5 independent experiments);***p<0.001; **p<0.01, Mann-Whitney test.


A 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 .

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A 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 .

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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 .

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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.


A , 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 .

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A, 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 ) .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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 .

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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.


A , 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 ) .

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A, 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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 .

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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.


B - 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 ) .

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B-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 .

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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 ) .

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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 ) .

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 .

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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.


A - 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 ) .

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A-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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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 .

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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.


A - 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 ) .

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A-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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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.


PD - 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 ) .

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PD-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 )

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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 .

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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 .

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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.


A . Characteristics of IGR39 / IGR37 and WM115 / WM266 . 4 melanoma cell lines ( Upper Panel ) . Pigmentation of melanoma cells pellets ( lower panel ) .

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A. 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 .

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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 .

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 .

D. Analysis of IGR39 and IGR37 speed of migration by time-lapse microscopy.


B . 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 .

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B. 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 .

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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 ) .

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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).


A . 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 ) .

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A. 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 .

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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 .

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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 .

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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.


A - 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 .

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A-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 ) .

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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).


A . Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB - 360 .

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A. 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 .

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 ) .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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.


Evaluation of expression by RNA - Seq and number of peptides required to detect an annotated protein by MS in M . pneumoniae . The plot represents the relationship between expression levels ( average expression from RNA - Seq data ) and number of possible unique tryptic peptides ( UTPs ) for two sets of studied proteins : detected ( blue dots ) and not detected ( orange dots ) by MS . B ) Evaluation of thresholds and artefactual signals in MS data . The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP , 1 UTP and 1NUTP , or ≥ 2 UTPs for three categories . Color code : annotated ( blue bars ) , putative new ( orange bars ) , and decoy set ( gray bars ) . C ) Number of SEPs detected by increasing the number of experiments . Color code is the same as in panels B . Each line represents the accumulated number of different SEPs detected ( Y - axis ) when combining 1 to 116 MS datasets ( X - axis ) from M . pneumoniae . Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets ( e . g . , x = 80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples ) .

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Evaluation of expression by RNA-Seq and number of peptides required to detect an annotated protein by MS in M. pneumoniae. The plot represents the relationship between expression levels (average expression from RNA-Seq data) and number of possible unique tryptic peptides (UTPs) for two sets of studied proteins: detected (blue dots) and not detected (orange dots) by MS. B) Evaluation of thresholds and artefactual signals in MS data. The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP, 1 UTP and 1NUTP, or ≥2 UTPs for three categories. Color code: annotated (blue bars), putative new (orange bars), and decoy set (gray bars). C) Number of SEPs detected by increasing the number of experiments. Color code is the same as in panels B. Each line represents the accumulated number of different SEPs detected (Y-axis) when combining 1 to 116 MS datasets (X-axis) from M. pneumoniae. Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets (e.g., x=80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples).


p53 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 .

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

p53 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Fluorescence 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Quantification 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 . )

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

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.)


Chk2 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 ) .

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 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

Chk2 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 .

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

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 ) .

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-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

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 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


Total p53 , p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics : pulsing p53 ( 10 Gy IR ) , transient p53 ( 10 Gy IR + BML - 277 , central lanes ) and sustained p53 ( 10 Gy IR + Nutlin - 3 , right lanes ) . See Figure 3 and Methods section for details . The relative change in p53 acetylation at K370 ( light green ) and K382 ( dark green ) was quantified from Western Blot and normalized to the abundance 3 h post IR . Means and propagated standard errors from 3 independent experiments are indicated . Acetylation increased over time in the context of sustained p53 . See also Appendix Fig S12 .

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

Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML-277, central lanes) and sustained p53 (10 Gy IR + Nutlin-3, right lanes). See Figure 3 and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western Blot and normalized to the abundance 3 h post IR. Means and propagated standard errors from 3 independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12.


The p53 - K370 methylase Smyd2 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated .

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The p53-K370 methylase Smyd2 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.


Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Smyd2 knockdown cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . 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 ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Fig 3 ) , while transcription rates remained unchanged .

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

Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Smyd2 knockdown cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). 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). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Fig 3), while transcription rates remained unchanged.


The p53 - K382 methylase Set8 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated .

The p53-K382 methylase Set8 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.


Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Set8 knock - down cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . 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 ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Figure 3 ) , while transcription rates remained unchanged .

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

Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Set8 knock-down cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). 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). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Figure 3), while transcription rates remained unchanged.


( A and B ) Endogenous FYCO1 binds to GST - LC3B . GST - LC3B bound to glutathione - Sepharose beads was incubated with HeLa cell lysate , and copurified proteins were identified by Coomassie blue staining and mass spectrometry ( A ) or immunoblotting with anti - FYCO1 antibody ( B ) .

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

(A and B) Endogenous FYCO1 binds to GST-LC3B. GST-LC3B bound to glutathione-Sepharose beads was incubated with HeLa cell lysate, and copurified proteins were identified by Coomassie blue staining and mass spectrometry (A) or immunoblotting with anti-FYCO1 antibody (B).


( C ) GST - LC3B but not GST - p62 or GST alone binds to FYCO1 in a GST pull - down assay . GST , GST - LC3B , or GST - p62 bound to glutathione - Sepharose beads was incubated with [ 35S ] methionine - labeled myc - FYCO1 . Formed protein complexes were isolated and visualized by autoradiography ( top ) or Coomassie blue staining ( bottom ) .

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

(C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione-Sepharose beads was incubated with [35S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom).


( D ) GFP - LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate . HeLa cells expressing GFP or GFP - LC3B were lysed and processed as in B . IP , immunoprecipitation ; MM , molecular mass ; WB , Western blot .

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

(D) GFP-LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate. HeLa cells expressing GFP or GFP-LC3B were lysed and processed as in B. IP, immunoprecipitation; MM, molecular mass; WB, Western blot.


( B ) The region of FYCO1 between aa 1 , 276 and 1 , 294 is essential and sufficient for the interaction with LC3B . GST or GST - LC3B were incubated with [ S35 ] methionine - labeled deletion mutants of FYCO1 and processed as in Fig . 1 C .

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 B-MEASURED_VAR O O O O O B-MEASURED_VAR 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) The region of FYCO1 between aa 1,276 and 1,294 is essential and sufficient for the interaction with LC3B. GST or GST-LC3B were incubated with [S35]methionine-labeled deletion mutants of FYCO1 and processed as in Fig. 1 C.


( C ) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells . HeLa cells expressing the indicated constructs were imaged by confocal microscopy . Insets show an enlarged field of interest .

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(C) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells. HeLa cells expressing the indicated constructs were imaged by confocal microscopy. Insets show an enlarged field of interest.


( D ) Both N - and C - terminal domains of LC3B are needed for efficient interaction with FYCO1 . GST , GST - LC3B , or its deletion mutants were incubated with [ S35 ] methionine - labeled myc - FYCO1 and processed as in Fig . 1 C . ARG , autoradiography ; CB , Coomassie blue .

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(D) Both N- and C-terminal domains of LC3B are needed for efficient interaction with FYCO1. GST, GST-LC3B, or its deletion mutants were incubated with [S35]methionine-labeled myc-FYCO1 and processed as in Fig. 1 C. ARG, autoradiography; CB, Coomassie blue.


( E ) The interaction between FYCO1 and LC3B is direct . GST or GST - LC3B was incubated with recombinant MBP - FYCO1863 - 1 , 478 . Protein complexes were isolated and visualized by immunoblotting with anti - GST or anti - MBP antibodies . WB , Western blot . Bars : ( C ) 10 µm ; ( C , insets ) 1 µm .

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(E) The interaction between FYCO1 and LC3B is direct. GST or GST-LC3B was incubated with recombinant MBP-FYCO1863-1,478. Protein complexes were isolated and visualized by immunoblotting with anti-GST or anti-MBP antibodies. WB, Western blot. Bars: (C) 10 µm; (C, insets) 1 µm.


( A ) Endogenous FYCO1 localizes to the cytosolic punctuated structures . HeLa cells cultured in complete growth medium ( left ) or in HBSS for 3 h ( right ) were fixed and stained with anti - FYCO1 antibody ( green ) and Draq5 ( blue ) .

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(A) Endogenous FYCO1 localizes to the cytosolic punctuated structures. HeLa cells cultured in complete growth medium (left) or in HBSS for 3 h (right) were fixed and stained with anti-FYCO1 antibody (green) and Draq5 (blue).


( B ) Overexpressed mCherry - FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells .

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(B) Overexpressed mCherry-FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells.


( C ) FYCO1 partially colocalizes with LC3B , LAMP1 , ATG5 , ATG16L , p62 , and HuntingtinQ64 and does not colocalize with EEA1 . HeLa cells or mouse embryonic fibroblasts ( for ATG16L staining ) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection . For p62 staining , HeLa cells were incubated with 0 . 2 µM BafA1 for 12 h before fixation .

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(C) FYCO1 partially colocalizes with LC3B, LAMP1, ATG5, ATG16L, p62, and HuntingtinQ64 and does not colocalize with EEA1. HeLa cells or mouse embryonic fibroblasts (for ATG16L staining) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection. For p62 staining, HeLa cells were incubated with 0.2 µM BafA1 for 12 h before fixation.


( D ) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures . HeLa cells cotransfected with GFP - FYCO1 , mCherry - LC3B , and myc - p62 were imaged by confocal microscopy 48 h after transfection . ( E and F ) FYCO1 - decorated structures are heterogenous in their luminal content .

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(D) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures. HeLa cells cotransfected with GFP-FYCO1, mCherry-LC3B, and myc-p62 were imaged by confocal microscopy 48 h after transfection. (E and F) FYCO1-decorated structures are heterogenous in their luminal content.


( E ) HeLa cells transfected with CFP - FYCO1 and mCherry - YFP - p62 were imaged 48 h after transfection .

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(E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection.


( F ) HeLa cells transfected with GFP - FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h in normal growth medium to stain late endocytic compartments ( left ) . Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647 - dextran ( 10 , 000 D ; right ) .

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(F) HeLa cells transfected with GFP-FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h in normal growth medium to stain late endocytic compartments (left). Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647-dextran (10,000 D; right).


( A ) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection . Insets show an enlarged field of interest .

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(A) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection. Insets show an enlarged field of interest.


( B - D ) Immunoelectron micrographs of HeLa cells transfected with GFP - FYCO1 and stained with anti - GFP polyclonal antibody ( 10 - nm gold particle ) .

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(B-D) Immunoelectron micrographs of HeLa cells transfected with GFP-FYCO1 and stained with anti-GFP polyclonal antibody (10-nm gold particle).


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( B ) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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(B) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles. HeLa cells were imaged 24 h after transfection with the indicated constructs.


( C ) FYCO1 can self - interact in a CC - dependent manner . Full - length GFP - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc - tagged FYCO1 in rabbit reticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - myc antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation .

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(C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.


( D ) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein - lipid overlay assay . PIP Strips were incubated with 1 - µg / ml solutions of MBP - FYVEFYCO1 , MBP - 2xFYVEFYCO1 , or MBP - FYCO1863 - 1 , 233 for 1 h , and bound proteins were detected by immunostaining with anti - MBP antibody . LPA , lysophosphatic acid ; LPC , lysophosphocholine ; PI , phosphatidylinositol ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine - 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

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(D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein-lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVEFYCO1, MBP-2xFYVEFYCO1, or MBP-FYCO1863-1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.


( E ) The construct containing a tandem fusion of two FYVE domains from FYCO1 ( 2xFYVEFYCO1 ) localizes to the nuclear envelope and ER in HeLa cells . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A and E ) Insets show an enlarged field of interest .

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(E) The construct containing a tandem fusion of two FYVE domains from FYCO1 (2xFYVEFYCO1) localizes to the nuclear envelope and ER in HeLa cells. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A and E) Insets show an enlarged field of interest.


( F ) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs ( left ) and PXp40phox ( right ) . HeLa cells were imaged 24 h after transfection with the indicated constructs . Bars , 10 µm .

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(F) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs (left) and PXp40phox (right). HeLa cells were imaged 24 h after transfection with the indicated constructs. Bars, 10 µm.


( A ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles . HeLa cells transiently transfected with FYCO1990 - 1 , 233 were imaged 24 h after transfection .

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(A) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles. HeLa cells transiently transfected with FYCO1990-1,233 were imaged 24 h after transfection.


( B ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 that can efficiently interact with the full - length FYCO1 . Full - length myc - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP - tagged FYCO1 in rabbitreticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - SDS - PAGE antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation .

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(B) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbitreticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-SDS-PAGE antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.


( C ) FYCO1990 - 1 , 233 strongly colocalizes with Rab7 , LysoTracker red , and Alexa Fluor 647 - dextran ( 10 , 000 D ) but only weakly with PXp40phox . HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h were imaged 24 h after transfection .

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(C) FYCO1990-1,233 strongly colocalizes with Rab7, LysoTracker red, and Alexa Fluor 647-dextran (10,000 D) but only weakly with PXp40phox. HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h were imaged 24 h after transfection.


( D ) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 ( FYCO1990 - 1 , 233 + 1 , 156 - 1 , 233 ) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A , C , and D ) Insets show an enlarged field of interest . Bars , 10 µm .

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(D) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 (FYCO1990-1,233+1,156-1,233) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A, C, and D) Insets show an enlarged field of interest. Bars, 10 µm.


( A ) Rab7Q67L recruits FYCO11 , 038 - 1 , 233 and FYCO11 , 091 - 1 , 233 but not FYCO11 , 156 - 1 , 233 to the cytosolic vesicles . HeLa cells transfected with GFP - FYCO11 , 038 - 1 , 233 ( top ) , GFP - FYCO11 , 091 - 1 , 233 ( bottom ) , or GFP - FYCO11 , 156 - 1 , 233 ( middle right ) with or without mCherry - Rab7Q67L were imaged 24 h after transfection . The graph shows the relative number of cells ± SD with GFP - FYCO11 , 038 - 1 , 233 recruited to intracellular membranes without and with cotransfected mCherry - Rab7Q67L .

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(A) Rab7Q67L recruits FYCO11,038-1,233 and FYCO11,091-1,233 but not FYCO11,156-1,233 to the cytosolic vesicles. HeLa cells transfected with GFP-FYCO11,038-1,233 (top), GFP-FYCO11,091-1,233 (bottom), or GFP-FYCO11,156-1,233 (middle right) with or without mCherry-Rab7Q67L were imaged 24 h after transfection. The graph shows the relative number of cells ± SD with GFP-FYCO11,038-1,233 recruited to intracellular membranes without and with cotransfected mCherry-Rab7Q67L.


( B ) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain ( FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 ) but not a 2xFYVE alone to the perinuclear vesicles . HeLa cells transfected with GFP - FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 or GFP - FYCO11 , 156 - 1 , 233 + 1 , 156 - 1 , 233 with or without mCherry - Rab7Q67L were imaged 24 h after transfection .

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(B) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain (FYCO11,091-1,233+1,156-1,233) but not a 2xFYVE alone to the perinuclear vesicles. HeLa cells transfected with GFP-FYCO11,091-1,233+1,156-1,233 or GFP-FYCO11,156-1,233+1,156-1,233 with or without mCherry-Rab7Q67L were imaged 24 h after transfection.


( C ) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate . HEK293 cells stably transfected with 3xFlag - FYCO1 were cotransfected with GTP - locked mutants of Rab5 , - 7 , - 11 , and - 24 and lysed 24 h after transfection , and protein complexes were immunoprecipitated with anti - Flag M2 agarose affinity gel . Bound proteins were eluted with Flag peptide , resolved by SDS - PAGE , and visualized by immunoblotting with anti - GFP and anti - Flag antibody . IP , immunoprecipitation ; WB , Western blot .

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(C) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate. HEK293 cells stably transfected with 3xFlag-FYCO1 were cotransfected with GTP-locked mutants of Rab5, -7, -11, and -24 and lysed 24 h after transfection, and protein complexes were immunoprecipitated with anti-Flag M2 agarose affinity gel. Bound proteins were eluted with Flag peptide, resolved by SDS-PAGE, and visualized by immunoblotting with anti-GFP and anti-Flag antibody. IP, immunoprecipitation; WB, Western blot.


( D ) Leucine - 1151 and tryptophan - 1152 are important for the recruitment of FYCO1 to Rab7 - containing vesicles . HeLa cells transfected with GFP - FYCO1990 - 1 , 233L1151A / W1152A with or without mCherry - Rab7Q67L were imaged 24 h after transfection . ( B and D ) Insets show an enlarged field of interest .

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(D) Leucine-1151 and tryptophan-1152 are important for the recruitment of FYCO1 to Rab7-containing vesicles. HeLa cells transfected with GFP-FYCO1990-1,233L1151A/W1152A with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (B and D) Insets show an enlarged field of interest.


( E ) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles . ( right ) HeLa cells cotransfected with GFP - RILP together with mCherry - FYCO1 and stained with Draq5 were imaged 24 h after transfection . ( left ) Relative number of HeLa cells cotransfected with mCherry - FYCO1 and the indicated constructs , in which FYCO1 is recruited to the intracellular vesicles . Error bars represent SD . Bars , 10 µm .

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(E) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles. (right) HeLa cells cotransfected with GFP-RILP together with mCherry-FYCO1 and stained with Draq5 were imaged 24 h after transfection. (left) Relative number of HeLa cells cotransfected with mCherry-FYCO1 and the indicated constructs, in which FYCO1 is recruited to the intracellular vesicles. Error bars represent SD. Bars, 10 µm.


( A ) Overexpressed FYCO1 is enriched in the cell periphery , close to the MT plus end . HeLa cells were imaged 48 h after transfection with mCherry - FYCO1 and GFP - tubulin .

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(A) Overexpressed FYCO1 is enriched in the cell periphery, close to the MT plus end. HeLa cells were imaged 48 h after transfection with mCherry-FYCO1 and GFP-tubulin.