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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

C-D, 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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O O O B-DISEASE O O O 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-ORGANISM O O O O

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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-DISEASE O O B-ORGANISM O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O

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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_LINE B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O

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 )

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O

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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-CELL_LINE O O O O O O O O B-DISEASE O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O

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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-DISEASE B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-DISEASE 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE O O O O O O O B-EXP_ASSAY O B-DISEASE O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-CELL_LINE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O 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. 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 .

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

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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 .

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-CELL_LINE O B-CELL_LINE O O O O B-CELL_LINE O B-CELL_LINE O B-DISEASE O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-CELL_LINE O B-CELL_LINE O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O

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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-ORGANISM O B-TISSUE O O O O O O O B-DISEASE O O O O B-DISEASE O O B-TISSUE O O O O B-DISEASE O O B-TISSUE O

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 ) .

O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-DISEASE O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

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 .

O O O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O 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-EXP_ASSAY O O O O O O O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O O O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O

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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. 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 .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-CELL_LINE O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. 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 .

O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. 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 ) .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD 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 B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O 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-EXP_ASSAY I-EXP_ASSAY 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD B-EXP_ASSAY O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD 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-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD 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-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD 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 .

O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

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 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

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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 ) .

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(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 ) .

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(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 .

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(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 .

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(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.


( B ) Peripheral enrichment of FYCO1 is dependent on intact MTs . HeLa cells were transfected with GFP - FYCO1 and cultured in normal medium ( left ) or treated with 5 µM colcemid ( middle ) or 5 µM latrunculin A ( right ) for 2 h .

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(B) Peripheral enrichment of FYCO1 is dependent on intact MTs. HeLa cells were transfected with GFP-FYCO1 and cultured in normal medium (left) or treated with 5 µM colcemid (middle) or 5 µM latrunculin A (right) for 2 h.


( C ) FYCO1 - decorated vesicles move along the MT tracks . HeLa cells transfected with mCherry - FYCO1 and GFP - tubulin were live imaged by confocal microscopy 48 h after transfection . Movements of FYCO1 - decorated structures were tracked with ImageJ MTrackJ plug - in ( National Institutes of Health ) and presented as 8 - min - long tracks in the overlay image .

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(C) FYCO1-decorated vesicles move along the MT tracks. HeLa cells transfected with mCherry-FYCO1 and GFP-tubulin were live imaged by confocal microscopy 48 h after transfection. Movements of FYCO1-decorated structures were tracked with ImageJ MTrackJ plug-in (National Institutes of Health) and presented as 8-min-long tracks in the overlay image.


( D ) Full - length FYCO1 but not the FYCO1 lacking aa 555 - 1 , 136 can redistribute Rab7 and ORP1L to the cell periphery . HeLa cells were transfected with the indicated constructs and stained with DRAQ5 .

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(D) Full-length FYCO1 but not the FYCO1 lacking aa 555-1,136 can redistribute Rab7 and ORP1L to the cell periphery. HeLa cells were transfected with the indicated constructs and stained with DRAQ5.


( E ) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1 - decorated structures . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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(E) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1-decorated structures. HeLa cells were imaged 24 h after transfection with the indicated constructs.


( A ) Western blot ( WB ) of cell lysates from HeLa cells stably expressing GFP - LC3 48 h after transient transfection with siRNA against FYCO1 ( siRNAs : A , Applied Biosystems ; D , Thermo Fisher Scientific ) . MM , molecular mass .

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(A) Western blot (WB) of cell lysates from HeLa cells stably expressing GFP-LC3 48 h after transient transfection with siRNA against FYCO1 (siRNAs: A, Applied Biosystems; D, Thermo Fisher Scientific). MM, molecular mass.


( B ) Representative images of HeLa cells with or without the perinuclear clusters of GFP - LC3 - positive vesicles ( right ) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 ( left ) . Error bars represent SDs based on three independent experiments .

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(B) Representative images of HeLa cells with or without the perinuclear clusters of GFP-LC3-positive vesicles (right) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 (left). Error bars represent SDs based on three independent experiments.


( C , D , and E ) The perinuclear clusters of GFP - LC3 - positive vesicles colocalize with mCherry - Rab7 but not with Golgi protein marker 58K or LysoTracker red . GFP - LC3 HeLa cells were transiently transfected with siRNA against FYCO1 ( Thermo Fisher Scientific ) 48 h before imaging or cotransfected with mCherry - Rab7 . Cells were either fixed and stained with anti - Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h . Insets show an enlarged field of interest . Bars , 10 µm .

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(C, D, and E) The perinuclear clusters of GFP-LC3-positive vesicles colocalize with mCherry-Rab7 but not with Golgi protein marker 58K or LysoTracker red. GFP-LC3 HeLa cells were transiently transfected with siRNA against FYCO1 (Thermo Fisher Scientific) 48 h before imaging or cotransfected with mCherry-Rab7. Cells were either fixed and stained with anti-Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h. Insets show an enlarged field of interest. Bars, 10 µm.


D , E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation ( D ) , and for molecules involved in V ( D ) J recombination and TCR signaling ( E ) . In D and E , dot color represents the scaled average expression of the specified gene across the various clusters , and dot size indicates the percentage of cells expressing the specified gene .

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D, E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation (D), and for molecules involved in V(D)J recombination and TCR signaling (E). In D and E, dot color represents the scaled average expression of the specified gene across the various clusters, and dot size indicates the percentage of cells expressing the specified gene.


C UMAP plots colored according to the expression level of the specified genes .

C UMAP plots colored according to the expression level of the specified genes.


D Dot plot showing the expression level of genes belonging to the γδ TCR - induced signature across the cell subsets corresponding to Dataset 2 . Based on the gene set that was specifically upregulated during the DPsmall → DPCD69 + / SP transition ( see Fig EV3B ) , it allowed to subdivide the γδ TCR - induced signature into a ' γδ TCR only ' signature and a ' αβ and γδ TCR shared '

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D Dot plot showing the expression level of genes belonging to the γδ TCR-induced signature across the cell subsets corresponding to Dataset 2. Based on the gene set that was specifically upregulated during the DPsmall → DPCD69+/SP transition (see Fig EV3B), it allowed to subdivide the γδ TCR-induced signature into a 'γδ TCR only' signature and a 'αβ and γδ TCR shared' signature


A , B UMAP plots of Dataset 3 which contains five WT ( DN3a , DN3b γδ + , DN4 γδ + , DN3b γδ - and DN4 γδ - ) and two Lat - / - ( DN3 γδ - and DN3 γδint ) scRNAseq datasets . The UMAP plot is colored according to sorted samples ( A ) , and to expression levels of the specified genes ( B ) .

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A, B UMAP plots of Dataset 3 which contains five WT (DN3a, DN3b γδ+, DN4 γδ+, DN3b γδ- and DN4 γδ-) and two Lat-/- (DN3 γδ- and DN3 γδint) scRNAseq datasets. The UMAP plot is colored according to sorted samples (A), and to expression levels of the specified genes (B).


A , B , C AUCell scores for the γδ TCR - induced signature ( A ) , γδ TCR only signature ( B ) and αβ and γδ TCR shared signature ( C ) are displayed on UMAP ( left ) and violin ( right ) plots of the 7 sorted subsets corresponding to Dataset 3 . As expected , AUCell scores for the 3 signatures were very low for DN3a cells , low for WT DN3b γδ - and WT DN4 γδ - cells , and high for WT DN3b γδ + and WT DN4 γδ + cells .

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A,B,C AUCell scores for the γδ TCR-induced signature (A), γδ TCR only signature (B) and αβ and γδ TCR shared signature (C) are displayed on UMAP (left) and violin (right) plots of the 7 sorted subsets corresponding to Dataset 3. As expected, AUCell scores for the 3 signatures were very low for DN3a cells, low for WT DN3b γδ- and WT DN4 γδ- cells, and high for WT DN3b γδ+ and WT DN4 γδ+ cells.


B Bar plot showing the percentage of cells in the G0 / G1 ( red bars ) , S ( blue bars ) and G2 / M ( green bars ) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3 .

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B Bar plot showing the percentage of cells in the G0/G1 (red bars), S (blue bars) and G2/M (green bars) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3.


( C ) High - performance thin - layer chromatography ( HPTLC ) profile of HeLa cells pulsed for 2 hours with [ 3H ] - sphingosine and chased for 24 hours . The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL .

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(C) High-performance thin-layer chromatography (HPTLC) profile of HeLa cells pulsed for 2 hours with [3H]-sphingosine and chased for 24 hours. The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL.


( A ) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [ 3H ] - sphingosine pulse - chase assay . GSL levels are expressed as fold changes with respect to control . CERT and FAPP2 knockdowns ( blue bars ) were used as controls .

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(A) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [3H] - sphingosine pulse-chase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls.


( D ) Control and GRASP55KO cells were processed for Cy3 - conjugated Shiga Toxin ( ShTxB ) and Alexa488 - conjugated Cholera Toxin ( ChTxB ) staining followed by flow cytometry analysis . Mean fluorescence intensity was measured and represented .

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(D) Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow cytometry analysis. Mean fluorescence intensity was measured and represented.


( C ) Control and GRASP55 KO cells were transfected for 16 hours with GCS - HA or LCS - HA and processed for cryoimmunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GM130 antibody ( 15 - nm gold particles ) in case of GCS - HA and anti - HA antibody ( 15 - nm gold particles ) and anti - GM130 antibody ( 10 - nm gold particles ) in case of LCS - HA . Representative images of the distribution of GCS - HA and LCS - HA are shown . Scale bar 200nm . Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling .

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(C) Control and GRASP55 KO cells were transfected for 16 hours with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling.


( A ) HeLa cells co - transfected with indicated HA tagged enzymes ( GCS , LCS , GM3S and Gb3S ) and GRASP55 - GFP were lysed , immunoprecipitated with anti - GFP antibody or control IgG and were analysed by western blotting for interaction by immunoblotting ( IB ) with anti - HA antibody . I represents 5 % on the input lysate , U 5 % of the unbound fraction and IP the immunoprecipitate . Red asterisks indicate IgG bands and arrow heads indicates the expected position of HA - tagged

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(A) HeLa cells co-transfected with indicated HA tagged enzymes (GCS, LCS, GM3S and Gb3S) and GRASP55-GFP were lysed, immunoprecipitated with anti-GFP antibody or control IgG and were analysed by western blotting for interaction by immunoblotting (IB) with anti-HA antibody. I represents 5% on the input lysate, U 5% of the unbound fraction and IP the immunoprecipitate. Red asterisks indicate IgG bands and arrow heads indicates the expected position of HA-tagged enzymes


( E ) Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS ( WT and Δ3C ) and indicated purified GST - tagged GRASP domain or SPR region of GRASP55 were incubated together and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by western blotting with an anti - GST tag antibody .

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(E) Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS (WT and Δ3C) and indicated purified GST-tagged GRASP domain or SPR region of GRASP55 were incubated together and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by western blotting with an anti-GST tag antibody.


( A - C ) Control and GRASP55 KO ( # 2 ) cells were transfected for 16 hours with the indicated HA - tagged enzymes and processed for cryo - immunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GM130 antibody ( 15 - nm gold particles ) . Representative images of the distribution of HA - tagged enzymes are shown . Red circles indicate GM130 labelling . Arrow heads represent the peri - Golgi vesicles . Scale bar 150 nm . Quantification of the distribution of enzymes in vesicles represented as normalized linear

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(A-C) Control and GRASP55 KO (#2) cells were transfected for 16 hours with the indicated HA-tagged enzymes and processed for cryo-immunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles indicate GM130 labelling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as normalized linear density


( F ) HeLa cells co - transfected with HA - tagged GCS and GRASP55 - GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GFP antibody ( 15 - nm gold particles ) . Representative images of the distribution of GCS and GRASP55 - GFP are shown . Red arrowhead indicates the clathrin - coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS - HA in TGN . Scale bar 150 nm . Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles .

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(F) HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead indicates the clathrin-coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles.


( A Control and GRASP55 KO clones were pre - treated with DMSO or BFA ( 5μg / ml ) for 30 min and SL output monitored by [ 3H ] - sphingosine pulse - chase assay ( 8 h chase ) . Total GSL levels were quantified and expressed as fold changes with respect to control ( A ) .

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(A Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). Total GSL levels were quantified and expressed as fold changes with respect to control (A).


B ) Control and GRASP55 KO clones were pre - treated with DMSO or BFA ( 5μg / ml ) for 30 min and SL output monitored by [ 3H ] - sphingosine pulse - chase assay ( 8 h chase ) . The GM / Gb ratio was calculated and represented ( B ) .

O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O

B) Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). The GM/Gb ratio was calculated and represented (B).


( F ) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO ( # 2 ) clone before subjecting to [ 3H ] - sphingosine pulse chase assay . GSLs were quantified and represented as fold change with respect to control .

O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O

(F) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO (#2) clone before subjecting to [3H] -sphingosine pulse chase assay. GSLs were quantified and represented as fold change with respect to control.


A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000 . Primary spheres were re - plated to evaluate secondary sphere formation . Bars indicate mean ± s . d . of 10 wells .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± s.d. of 10 wells.