Figure 2Scanning electron microscopy of single bacteria on a cellulose filter . Scale bars : 500 nm . Two representative immunofluorescence labelling of TFP . Scale bar : 1 µm . Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody . Black dots indicate all the individual de novo generated PilE mutants that were characterized . Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region . Solid lines indicate 95 % confidence interval . Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants . Mean values ± SEM are indicated for each strain . N ≥ 3 independent experiments . Paired t - test . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm.Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).


Figure 3Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody . Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated ( magenta ) . In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling . Scale bar : 2 µm . Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported . Each dot represents values for a PilE point mutant relative to the SB strain . Hypo - aggregative mutants are highlighted in orange while hyper - aggregative mutants are in light blue . solid lines indicate the 95 % confidence interval . Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot . PilX levels were normalized using PilE as a reference and normalized to pilESB values . Mean ratio ± SEM is indicated . N = 3 independent experiments . Paired t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval.Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.


Figure 4Early adhesion of pilE mutants as a function of aggregation . Values were normalized to that of pilESB . Each dot represents a pilE mutant and the pilV , pilD , pilX and pilC1 reference mutants . Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple . A blown - up view of the zone of interest ( lower left quarter ) is presented on the right panel . The shaded area highlights the absence of significant adhesion . Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB . Piliation length of a selection of mutants determined by immunofluorescence . Average pilus length ± SEM is indicated . N = 6 experiments with at least 50 bacteria analyzed in each experiment . Paired t - test . Number of pili per bacteria ± SEM determined as in ( E ) . Paired t - test . p < 0 . 05 ( * ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion.Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB.Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test. Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*).


Figure 5Time - lapse of a cell - adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes . Initiation of flow corresponds to t = 0 . Scale bar : 1 µm . Spinning - disk confocal immunofluorescence of pili stained with both SM1 ( magenta ) and F10 ( green ) . The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light . In each field of view , a substrate - and a cell - bound pilus are shown . Upper panels : z - projection from the stacks in the xy plane ; bottom panels : side view in the z plane . Scale bar : 2 µm . White arrows correspond to SM1 staining at the tip of pili . Quantification of adhesion after infection of HUVEC cells for 30 minutes , expressed as the number of adherent bacteria over the number of cell nuclei . Prior to infection , bacteria were incubated at 37 ° C with or without antibody or nanobody for 15 minutes . Mean values ± SEM are indicated for each strain . N = 5 independent experiments . Paired t - test . p < 0 . 05 ( * ) .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes. Initiation of flow corresponds to t=0. Scale bar: 1 µm.Spinning-disk confocal immunofluorescence of pili stained with both SM1 (magenta) and F10 (green). The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light. In each field of view, a substrate- and a cell-bound pilus are shown. Upper panels: z-projection from the stacks in the xy plane; bottom panels: side view in the z plane. Scale bar: 2 µm. White arrows correspond to SM1 staining at the tip of pili. Quantification of adhesion after infection of HUVEC cells for 30 minutes, expressed as the number of adherent bacteria over the number of cell nuclei. Prior to infection, bacteria were incubated at 37°C with or without antibody or nanobody for 15 minutes. Mean values ± SEM are indicated for each strain. N=5 independent experiments. Paired t-test. p<0.05 (*).


Figure 1 ( A ) miR - 515 - 5p expression increases the area of the tubulin cytoskeleton . Tubulin ( green ) and cell nucleus ( blue ) . Objective x20 . Bar : 200 µm . ( B - C ) miR - 515 - 5p inhibits random ( B ) and directed ( C ) cell migration . The indicated cell lines were transfected with miR - 515 - 5p for 48 h before time - lapse imaging was performed for 18 h ( A ) or transwell migration assays were performed for 9 h ( C ) . ( B - left panel ) Plots show overlays of representative trajectories travelled . ( B - right panel ) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition . ( C - left panel ) Representative field of view from the bottom of the transwell chambers . Scale bar is 20 µm . ( C - right panel ) Data represent percentage of the average number migratory cells per field ( n = 5 fields per condition ) . Data are mean of three experiments ± SEM . ( B - C ) P values were calculated by t - test between miRNA ( 515 - 5p ) values and their respective non - targeting control miR ( NC ) values ( * * * , P < 0 . 001 ) . All data shown are representative of experiments performed at least in triplicate .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) miR-515-5p expression increases the area of the tubulin cytoskeleton. Tubulin (green) and cell nucleus (blue). Objective x20. Bar: 200 µm.(B-C) miR-515-5p inhibits random (B) and directed (C) cell migration. The indicated cell lines were transfected with miR-515-5p for 48 h before time-lapse imaging was performed for 18 h (A) or transwell migration assays were performed for 9 h (C). (B-left panel) Plots show overlays of representative trajectories travelled. (B-right panel) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition. (C-left panel) Representative field of view from the bottom of the transwell chambers. Scale bar is 20 µm. (C-right panel) Data represent percentage of the average number migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (B-C) P values were calculated by t-test between miRNA (515-5p) values and their respective non-targeting control miR (NC) values (***, P<0.001). All data shown are representative of experiments performed at least in triplicate.


Figure 2 ( A ) RNA - seq of MCF7 and MDA - MB - 231 transfected with miR - 515 - 5p revealed the down - regulation of five transcripts , NRAS , FZD4 , CDC42BPA , PIK3C2B and MARK4 . ( B - D ) The effect of miR - 515 - 5p overexpression on NRAS , FZD4 , CDC42BPA , PIK3C2B and MARK4 mRNA levels in MCF7 ( B ) , MDA - MB - 231 ( C ) , A549 and H1299 ( D ) was determined by qPCR 48 h following transfection of the indicated cell line with non - targeting control miR ( NC ) or miR - 515 - 5p . Data is displayed as the normalised mean ± SEM . P values were calculated by t - test between miRNA conditions and their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) . ( E - F ) miR - 515 - 5p directly interacts with NRAS , MARK4 and PI3KC2B ' s 3 ' UTR . Relative luciferase activity levels were measured 24 h after co - transfection of MCF - 7 ( E ) and MDA - MB - 231 ( F ) with 3 ' UTR - luciferase reporter constructs and either with miR - 515 - 5p or miR NC . Data shown are normalised mean of three independent experiments ± SEM . ( B - F ) P values were calculated by t - test between miRNA conditions and their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) RNA-seq of MCF7 and MDA-MB-231 transfected with miR-515-5p revealed the down-regulation of five transcripts, NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4.(B-D) The effect of miR-515-5p overexpression on NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNA levels in MCF7 (B), MDA-MB-231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non-targeting control miR (NC) or miR-515-5p. Data is displayed as the normalised mean ± SEM. P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).(E-F) miR-515-5p directly interacts with NRAS, MARK4 and PI3KC2B's 3'UTR. Relative luciferase activity levels were measured 24 h after co-transfection of MCF-7 (E) and MDA-MB-231 (F) with 3'UTR-luciferase reporter constructs and either with miR-515-5p or miR NC. Data shown are normalised mean of three independent experiments ± SEM. (B-F) P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).


Figure 3MARK4 siRNA - mediated silencing increases the tubulin cytoskeletal area ( A ) . ( A ) Data shown are representative of experiments performed in triplicate . Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions . MARK4 siRNA - mediated silencing reduces random ( B ) migration in MDA - MB - 231 ( MDA ) , A549 and H1299 cells . Cells were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before time - lapse imaging was performed for 18 h ( B ) . ( B - top panel ) Plots show overlays of representative trajectories described . ( B - bottom panel ) The migration distance was quantified and represented as the normalised mean ± SEM . ( C - top panel ) Representative field of view from the bottom side of the transwell membrane . ( C - bottom panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the siRNA - transfected and the respective control condition ( * * * P < 0 . 001 ) . MARK4 siRNA - mediated silencing reduces directed ( C ) migration in MDA - MB - 231 ( MDA ) , A549 and H1299 cells . ( C - top panel ) . Cells were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before transwell assay for 9h ( C ) . Representative field of view from the bottom side of the transwell membrane . ( C - bottom panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the siRNA - transfected and the respective control condition ( * * * P < 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3MARK4 siRNA-mediated silencing increases the tubulin cytoskeletal area (A). (A) Data shown are representative of experiments performed in triplicate. Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions.MARK4 siRNA-mediated silencing reduces random (B) migration in MDA-MB-231 (MDA), A549 and H1299 cells.Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before time-lapse imaging was performed for 18 h (B). (B-top panel) Plots show overlays of representative trajectories described. (B-bottom panel) The migration distance was quantified and represented as the normalised mean ± SEM. (C-top panel) Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).MARK4 siRNA-mediated silencing reduces directed (C) migration in MDA-MB-231 (MDA), A549 and H1299 cells.(C-top panel). Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before transwell assay for 9h (C). Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).


Figure 4 ( A - B ) The indicated cell lines were transfected with either miR - 515 - 5p or a non - targeting miR ( miR NC ) together with a MARK4 expressing or an empty vector ( EV ) plasmid DNA . MARK4 over - expression rescues the inhibition by miR - 515 - 5p of random ( A ) and directed ( B ) migration of the indicated cell lines . ( A - left panel ) Plots show overlays of representative trajectories described for MDA - MB - 231 cells ( n = 30 ) . ( A - right panel ) The migration distance was quantified and represented as the normalised mean ± SEM . ( B - left panel ) Representative field of view from the bottom side of the transwell membrane for MDA - MB - 231 . ( B - right panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the transfected and the respective control condition ( EV + miR - NC ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-B) The indicated cell lines were transfected with either miR-515-5p or a non-targeting miR (miR NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 over-expression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A-left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n=30). (A-right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B-left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B-right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*,P<0.05; **,P<0.01; ***,P<0.001).


Figure 5 ( A - B ) Overall survival of ER - negative metastatic breast cancer ( n = 226 ) ( A ) and lung adenocarcinoma ( n = 719 ) ( B ) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier - plotter [ 25 ] . ( C ) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression . Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie - chart . ( D - F ) MARK4 protein expression was determined by histoimmunochemistry in ( D - E ) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or ( F ) a home - made tissue microarray comprising 100 matched cases of primary and metastatic tumors . ( D - E ) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples ( D ) or in lung tumors according to their histological subtypes ( E ) is represented . ( F ) The percentage of cases with increased , decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-B) Overall survival of ER-negative metastatic breast cancer (n=226) (A) and lung adenocarcinoma (n=719) (B) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier-plotter [25].(C) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression. Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie-chart.(D-F) MARK4 protein expression was determined by histoimmunochemistry in (D-E) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or (F) a home-made tissue microarray comprising 100 matched cases of primary and metastatic tumors. (D-E) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples (D) or in lung tumors according to their histological subtypes (E) is represented. (F) The percentage of cases with increased, decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented.


Figure 6 ( A - D ) Low expression of miR - 515 - 5p is linked to survival in both Lung and Breast cancer . ( A ) Survival analysis of miR - 515 - 5p from 195 lung adenocarcinoma samples from the TCGA . ( B ) Survival analysis of miR - 515 - p from 34 lung squamous cell carcinoma from the TCGA . ( C ) Survival analysis of miR - 515 - 5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study ( GSE37405 ) . ( D ) Survival analysis of miR - 515 - 5p from 33 Breast cancer from the GSE7405 Breast study . Analysis and plot were obtained with SurvMicro http : / / bioinformatica . mty . itesm . mx : 8080 / Biomatec / Survmicro . jsp for ( A - C ) and with MIRUMIR for ( D ) . ( E ) Comparison of miR - 515 - 5p expression in 18 primary and matched metastatic lesions from breast cancer patients . miR - 515 - 5p levels were quantified by TaqMan qRT - PCR . Absolute values were normalized to u6 snRNA values and displayed as a dot - plot . Bar ; Mean , Whiskers ; SEM . ( F ) MDA - MB - 231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [ 22 ] . miR - 515 - 5p and MARK4 levels were then determined in the primary and indicated secondary lesions . Data are average ± SEM of technical triplicates . ( G ) Luciferase - expressing A549 cells overexpressing miR - 515 - 5p or a non - targeting control ( miR - NC ) were injected in the tail - vein of SCID mice and the development of tumor burden monitored by whole animal - imaging in response to luciferin administration at week 1 and 4 following injection . ( G - left panel ) Representative imaging of the same animals for the indicated conditions at week 1 and 4 . ( G - Right panel ) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1 . Data represented are representative of three independent experiments . ( E - G ) P values were calculated by t - test ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-D) Low expression of miR-515-5p is linked to survival in both Lung and Breast cancer. (A) Survival analysis of miR-515-5p from 195 lung adenocarcinoma samples from the TCGA. (B) Survival analysis of miR-515-p from 34 lung squamous cell carcinoma from the TCGA. (C) Survival analysis of miR-515-5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study (GSE37405). (D) Survival analysis of miR-515-5p from 33 Breast cancer from the GSE7405 Breast study. Analysis and plot were obtained with SurvMicro http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp for (A-C) and with MIRUMIR for (D).(E) Comparison of miR-515-5p expression in 18 primary and matched metastatic lesions from breast cancer patients. miR-515-5p levels were quantified by TaqMan qRT-PCR. Absolute values were normalized to u6 snRNA values and displayed as a dot-plot. Bar; Mean, Whiskers; SEM.(F) MDA-MB-231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [22]. miR-515-5p and MARK4 levels were then determined in the primary and indicated secondary lesions. Data are average ± SEM of technical triplicates.(G) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).


Figure 7A549 - Luc cells transfected either with miR - 515 - 5p precursor ( miR ) , siRNAs to MARK4 ( siMARK4 ) or the corresponding control sequences ( C and NT , respectively ) were injected in Scid mice through the tail vein . 4 weeks later , the mice were sacrificed , their lungs extracted and fixed prior to staining for Luciferase expression . ( A ) Representative pictures showing the presence of A549 - Luc cells in the lungs of NT mice either as single cells ( upper panel ) or as a tumour mass ( lower panel ) . ( B ) Percentage of the area covered by tumour cells on randomly observed fields of views . Quantification was performed using Photoshop CS4 . Each spot corresponds to quantification of an individual 1 mm x 1 . 5 mm field . Transversal bar ; Median . Statistics : Student t - test . * , p ≤ 0 . 05 .

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Figure 7A549-Luc cells transfected either with miR-515-5p precursor (miR), siRNAs to MARK4 (siMARK4) or the corresponding control sequences (C and NT, respectively) were injected in Scid mice through the tail vein. 4 weeks later, the mice were sacrificed, their lungs extracted and fixed prior to staining for Luciferase expression. (A) Representative pictures showing the presence of A549-Luc cells in the lungs of NT mice either as single cells (upper panel) or as a tumour mass (lower panel). (B) Percentage of the area covered by tumour cells on randomly observed fields of views. Quantification was performed using Photoshop CS4. Each spot corresponds to quantification of an individual 1 mm x 1.5 mm field. Transversal bar; Median. Statistics: Student t-test. *, p≤0.05.


Figure 2 ( C ) Representative IHC images of CD3 + T cells and Iba1 + macrophages / MG in the isotype and αCD4 + αCD8 group . Scale bar ; 100 μm . ( D ) Quantification of Iba1 + and CD3 + cells in IHC sections of BrM from the isotype ( n = 9 ) and αCD4 + αCD8 group ( n = 7 ) . ( F ) Kaplan - Meier curves depict BrM free survival of mice in the isotype and αCD4 + αCD8 group ( Isotype n = 19 , αCD4 + αCD8 n = 19 ) . ( H ) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL - CD4 and TIL - CD8 compared to blood lymphocytes . Values display the average per group based on vst values ( BL - CD4 or BL - CD8 with n = 3 for each group ) vs . BrM - associated TIL - CD4 or TIL - CD8 ( n = 3 for each group ) .

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Figure 2(C) Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4+αCD8 group. Scale bar; 100 μm. (D) Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n=9) and αCD4+αCD8 group (n=7).(F) Kaplan-Meier curves depict BrM free survival of mice in the isotype and αCD4+αCD8 group (Isotype n=19, αCD4+αCD8 n=19).(H) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared to blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n=3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n=3 for each group).


Figure 3 ( A ) Surface expression of PD - 1 in vivo in 99LN - BrM quantified by flow cytometry ( n = 3 ) . ( B ) PD - L1 in 99LN BrM quantified by flow cytometry ( n = 3 ) . ( C ) Flow cytometric analysis of PD - L1 in tumor - free brain and 99LN - BrM ( n = 3 ) . ( D ) Flow cytometric analysis of PD - L1 expression on 99LN - BrM associated my myeloid cell types . ( n = 6 ) . ( H ) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN - BrM and representative IF images of multiplexed histology . Scale bar ; 100 μm .

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Figure 3(A) Surface expression of PD-1 in vivo in 99LN-BrM quantified by flow cytometry (n=3). (B) PD-L1 in 99LN BrM quantified by flow cytometry (n=3). (C) Flow cytometric analysis of PD-L1 in tumor-free brain and 99LN-BrM (n=3). (D) Flow cytometric analysis of PD-L1 expression on 99LN-BrM associated my myeloid cell types. (n=6).(H) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN-BrM and representative IF images of multiplexed histology. Scale bar; 100 μm.


Figure 4 ( C ) Quantification of Iba1 + macrophages in IHC sections of 99LN - BrM at trial endpoint ( Ctrl n = 27 , WBRT n = 18 ) . ( D ) Representative IF images of 99LN - BrM stained for the macrophage marker Iba1 ( red ) and the MG marker Tmem119 ( white ) . Higher magnification images present areas in the peri - tumor region and in the tumor core . DAPI was used as nuclear counterstain . Scale bar ; 100 µm . ( G ) Quantification of CD11c + MHCII + immune cells by flow cytometry in 99LN - BrM with and without WBRT . ( H , I ) Relative proportion of cDC1 and cDC2 in 99LN - BrM with and without WBRT . ( J ) cDC1 : cDC2 ratio in 99LN - BrM based on flow cytometry data ( H + I ) . ( K ) Representative IHC images of CD3 + , FoxP3 + and CD8 + T cells in 99LN - BrM sections with and without WBRT . Scale bar ; 100 µm . ( L ) Quantification of CD3 + cells in IHC sections of 99LN - BrM . ( Ctrl n = 26 , WBRT n = 18 ) . ( M ) FoxP3 : CD3 ratio in IHC sections of 99LN - BrM . ( Ctrl n = 25 , WBRT n = 18 ) . ( N ) CD8 : CD3 ratio in IHC sections of 99LN - BrM . ( Ctrl n = 25 , WBRT n = 18 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C) Quantification of Iba1+ macrophages in IHC sections of 99LN-BrM at trial endpoint (Ctrl n=27, WBRT n=18). (D) Representative IF images of 99LN-BrM stained for the macrophage marker Iba1 (red) and the MG marker Tmem119 (white). Higher magnification images present areas in the peri-tumor region and in the tumor core. DAPI was used as nuclear counterstain. Scale bar; 100 µm.(G) Quantification of CD11c+MHCII+ immune cells by flow cytometry in 99LN-BrM with and without WBRT. (H, I) Relative proportion of cDC1 and cDC2 in 99LN-BrM with and without WBRT. (J) cDC1:cDC2 ratio in 99LN-BrM based on flow cytometry data (H+I). (K) Representative IHC images of CD3+, FoxP3+ and CD8+ T cells in 99LN-BrM sections with and without WBRT. Scale bar; 100 µm. (L) Quantification of CD3+ cells in IHC sections of 99LN-BrM. (Ctrl n=26, WBRT n=18). (M) FoxP3:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18). (N) CD8:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18).


Figure 5 ( B ) Percent T cells of estimated cells in BrM and CLN of mice from control or WBRT group .

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Figure 5(B) Percent T cells of estimated cells in BrM and CLN of mice from control or WBRT group.


Figure 6 ( B ) Representative MRI images of 99LN - BrM from mice at different time points after treatment start . ( G ) Kaplan - Meier curves show the percentage of symptom - free mice .

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Figure 6(B) Representative MRI images of 99LN-BrM from mice at different time points after treatment start.(G) Kaplan-Meier curves show the percentage of symptom-free mice.


Figure 7 ( K ) Relative Gzmb and IFNγ protein level on CD4 + and CD8 + T cells cultivated with different cell types , with or without aPD - 1 and unstimulated or stimulated with tumor conditioned media ( Cond . ) ( n = 4 for conditions including BMDM , n = 5 for all other conditions ) .

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Figure 7(K) Relative Gzmb and IFNγ protein level on CD4+ and CD8+ T cells cultivated with different cell types, with or without aPD-1 and unstimulated or stimulated with tumor conditioned media (Cond.) (n=4 for conditions including BMDM, n=5 for all other conditions).


Figure 1A . Frequencies and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen and peripheral LN of naïve non - immunized mice . B . Total numbers ( mean + SEM ) of CD11b + cells expressing IL - 1β shown in ( A ) and restimulated with indicated stimuli for 4 h . C . Quantification ( mean + SEM ) of myeloid cell populations expressing IL - 1β , isolated from LN and restimulated with GM - CSF shown in ( B ) . D . Frequencies and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen of immunized mice . E . Analysis of IL - 1R1 expression by CD11b + cells isolated from dLN of mice treated as in ( D ) . Data are representative FACS histogram overlays , gated on VD - CD11b + cells with mean frequencies per group . F . Quantification ( mean + SEM ) of IL - 1β expression by CD11b + cells shown in ( E ) . G . Analysis of IL - 1β expressing CD11b + cells shown in ( E ) . Data are representative FACS plots , gated on VD - CD11b + IL - 1β + cells with mean frequencies per group . H . Quantification ( mean + SEM ) of myeloid cell populations expressing IL - 1β shown in ( G ) .

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Figure 1A. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen and peripheral LN of naïve non-immunized mice.B. Total numbers (mean +SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h.C. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β, isolated from LN and restimulated with GM-CSF shown in (B).D. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen of immunized mice.E. Analysis of IL-1R1 expression by CD11b+cells isolated from dLN of mice treated as in (D). Data are representative FACS histogram overlays, gated on VD-CD11b+cells with mean frequencies per group.F. Quantification (mean +SEM) of IL-1β expression by CD11b+ cells shown in (E).G. Analysis of IL-1β expressing CD11b+cells shown in (E). Data are representative FACS plots, gated on VD- CD11b+IL-1β+cells with mean frequencies per group.H. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β shown in (G).


Figure 2A - D . Analysis of IL - 1R1 expression by different populations of murine ( A ) CD4 T cells and ( B ) Th17 cells isolated from the dLN 9 days after immunization ; and ( C ) CD4T cells and ( D ) Th17 cells isolated from the CNS at the peak of EAE . Cells ( A - D ) were restimulated in the presence of MOG for 6 h . Data consist of n = 4 wild type mice immunized with MOG / CFA / PTx . Data ( A , C ) are representative FACS histogram overlay of VD - TCRβ + CD4 + cells gated on IL - 17A + , IL - 17A - IFNγ + ( IFNγ + ) , IL - 17A - GM - CSF + ( GM - CSF + ) or IL - 17A - FoxP3 + ( FoxP3 + ) cells ( also see Appendix Fig S1 ) and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results . E - F . Analysis of IL - 1R1 expression by different populations of human ( E ) CD4 T cells and ( F ) Th17 cells isolated from the peripheral blood . Cells ( E - F ) were restimulated with PMA / Ionomycin for 4 h . Data consist of PBMC isolated from n = 4 healthy individuals . Data ( E ) are FACS plots of cells gated on CD14 - CD3 + CD4 + cells with mean frequencies per group and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D. Analysis of IL-1R1 expression by different populations of murine (A) CD4 T cells and (B) Th17 cells isolated from the dLN 9 days after immunization; and (C) CD4T cells and (D) Th17 cells isolated from the CNS at the peak of EAE.Cells (A-D) were restimulated in the presence of MOG for 6 h. Data consist of n=4 wild type mice immunized with MOG/CFA/PTx.Data (A, C) are representative FACS histogram overlay of VD-TCRβ+CD4+ cells gated on IL-17A+, IL-17A-IFNγ+ (IFNγ+), IL-17A-GM-CSF+ (GM-CSF+) or IL-17A-FoxP3+ (FoxP3+) cells (also see Appendix Fig S1) and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.Data (E) are FACS plots of cells gated on CD14-CD3+CD4+cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.


Figure 3A . Analysis of cytokine expression by CD4T cells isolated from the spleen . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4T cells per group ± SEM , and ( lower row ) with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results . B . Frequencies and total numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the dLN . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results . C - D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + Th17 cells isolated from the spleen ( C ) and dLN ( D ) . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Analysis of cytokine expression by CD4T cells isolated from the spleen. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.B. Frequencies and total numbers (mean +SEM) of MOG-specific Th17 cells isolated from the dLN.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.C-D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+ Th17 cells isolated from the spleen (C) and dLN (D).Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


Figure 4A . Analysis of CD4T cells isolated from the dLN and restimulated with PMA / Ionomycin for 4 h . Data are representative FACS plots , gated on VD - CD4 + cells with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results . B . Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4T cells per group ± SEM , and ( lower row ) with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results . C . Total cell numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the spleen , shown in ( B , upper row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results . D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + IFNγ + / - Th17 cells isolated from the spleen , shown in ( B , lower row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Analysis of CD4T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+cells with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.B. Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.C. Total cell numbers (mean +SEM) of MOG-specific Th17 cells isolated from the spleen, shown in (B, upper row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+IFNγ+/- Th17 cells isolated from the spleen, shown in (B, lower row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


Figure 5A - C . ( A ) Mean , ( B ) maximal EAE clinical scores and ( C ) area under the curve ( AUC ) of mice immunized with MOG / CFA / PTx . Data shown as ( A ) mean ( + SEM ) , ( B , C ) individual plots with mean and are combined from at least four independent experiments and consist of n = 41 WT , n = 24 IL - 1R1ΔT , n = 18 IL - 1R1 - / - mice . * p < 0 . 05 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Black ( * ) signs in ( A ) represent statistical analysis of IL - 1R1ΔT vs WT and blue ( * ) of IL - 1R1ΔT vs IL - 1R1 - / - groups . D - E . ( D ) Maximal EAE clinical scores ( individual plots with mean ) of selected miceD - E . ( E ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . F - H . Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in ( D ) . Data ( F ) are representative FACS plots , gated on VD - CD90 + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4 T cells per group ± SEM , and ( lower row ) with mean frequencies per group . ( G ) Mean fluorescent intensity ( mean + SEM ) of IL - 17A staining by MOG - specific Th17 cells , shown in ( F , upper row ) . ( H ) Quantification of MOG - specific GM - CSF + Th17 cells ( mean + SEM ) shown in ( F , lower row ) . I - K . Analysis of Treg cells isolated from mice shown in ( D ) . ( I ) Total Treg cell numbers ( mean + SEM ) isolated from CNS and dLN . ( J ) Ratio of Treg and Th17cells isolated from the CNS . Data are representative FACS plots , gated on VD - CD90 + CD4 + cells with mean frequencies per group . ( K ) Quantification of Treg / Th17 cell ratio ( mean + SEM ) within CNS and dLN . Cells were restimulated with MOG for 6 h and ( I - N ) in the presence of PMA / Ionomycin for 4 h . L - N . Analysis of CCR6 expression by CD4 T cells isolated from the dLN and CNS of EAE diseased mice . Data ( L ) are representative FACS histogram overlays of ( black ) control , ( blue ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A + cells ; and of ( black filled ) control , ( blue filled ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A - cells . ( M ) Mean fluorescent intensity ( mean + SEM ) of CCR6 staining by CD4 T cells , shown in ( L ) . ( N ) Mean fluorescent intensity ( mean + SEM ) of CCR6 staining by GM - CSF + and GM - CSF - Th17 cells isolated from CNS , shown in ( L ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-C. (A) Mean, (B) maximal EAE clinical scores and (C) area under the curve (AUC) of mice immunized with MOG/CFA/PTx. Data shown as (A) mean (+SEM), (B, C) individual plots with mean and are combined from at least four independent experiments and consist of n=41 WT, n=24 IL-1R1ΔT, n=18 IL-1R1-/-mice. *p < 0.05, ***p < 0.001; two-tailed unpaired t-test. Black (*) signs in (A) represent statistical analysis of IL-1R1ΔT vs WT and blue (*) of IL-1R1ΔT vs IL-1R1-/- groups.D-E. (D) Maximal EAE clinical scores (individual plots with mean) of selected miceD-E. (E) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.F-H. Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in (D). Data (F) are representative FACS plots, gated on VD-CD90+CD4+CD44+cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group. (G) Mean fluorescent intensity (mean +SEM) of IL-17A staining by MOG-specific Th17 cells, shown in (F, upper row). (H) Quantification of MOG-specific GM-CSF+ Th17 cells (mean +SEM) shown in (F, lower row).I-K. Analysis of Treg cells isolated from mice shown in (D). (I) Total Treg cell numbers (mean +SEM) isolated from CNS and dLN. (J) Ratio of Treg and Th17cells isolated from the CNS. Data are representative FACS plots, gated on VD-CD90+CD4+cells with mean frequencies per group. (K) Quantification of Treg/Th17 cell ratio (mean +SEM) within CNS and dLN.Cells were restimulated with MOG for 6 h and (I-N) in the presence of PMA/Ionomycin for 4 h.L-N. Analysis of CCR6 expression by CD4 T cells isolated from the dLN and CNS of EAE diseased mice. Data (L) are representative FACS histogram overlays of (black) control, (blue) IL-1R1ΔTcells, gated on CD3+CD4+IL-17A+cells; and of (black filled) control, (blue filled) IL-1R1ΔTcells, gated on CD3+CD4+IL-17A-cells. (M) Mean fluorescent intensity (mean +SEM) of CCR6 staining by CD4 T cells, shown in (L). (N) Mean fluorescent intensity (mean +SEM) of CCR6 staining by GM-CSF+ and GM-CSF-Th17 cells isolated from CNS, shown in (L).


Figure 6A - C . Analysis of ( A ) IL - 17A expression by wild type CD4T cells , followed with the analysis of ( B ) GM - CSF and ( C ) GM - CSF and IFNγ co - expression by Th17 cells . D - G . Analysis of cytokine expression by cells isolated from WT ( black ) , IL - 1R1ΔT ( blue ) and IL - 1R1 - / - ( red ) mice and cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + cells with mean frequencies per group . Cells ( A - G ) were isolated from dLN and the spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis . Data consist of n = 4 of each genotype . D - G . Analysis of cytokine expression by cells isolated from WT ( black ) , IL - 1R1ΔT ( blue ) and IL - 1R1 - / - ( red ) mice and cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + cells with mean frequencies per group . Quantification ( mean + SEM ) of ( E ) MOG - specific Th17 cells , ( F ) GM - CSF + Th17 cells and ( G ) GM - CSF + IFNγ + Th17 cells . Cells ( A - G ) were isolated from dLN and the spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis . Data consist of n = 4 of each genotype . H - I . ( H ) Mean EAE clinical scores ( + SEM ) of Rag1 - / - mice which received 1 x 105 IL - 17A + cells of indicated genotypes and ( I ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results . H - I . Rag1 - / - mice which received 1 x 105 IL - 17A + cells of indicated genotypes and ( I ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results . J - L . Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in ( H ) and restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + cells ( upper row ) with mean frequencies ± SEM . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results . J - L . Analysis of cytokine expression by CD4T cells isolated from CNS of mice shown in ( H ) and restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + cells ( upper row ) with mean frequencies ± SEM . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-C. Analysis of (A) IL-17A expression by wild type CD4T cells, followed with the analysis of (B) GM-CSF and (C) GM-CSF and IFNγ co-expression by Th17 cells.D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+cells with mean frequencies per group.Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+cells with mean frequencies per group. Quantification (mean +SEM) of (E) MOG-specific Th17 cells, (F) GM-CSF+Th17 cells and (G) GM-CSF+IFNγ+Th17 cells.Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.H-I. (H) Mean EAE clinical scores (+SEM) of Rag1-/- mice which received 1 x 105 IL-17A+cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.H-I. Rag1-/- mice which received 1 x 105 IL-17A+cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.J-L. Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+cells (upper row) with mean frequencies ± SEM.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.J-L. Analysis of cytokine expression by CD4T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+cells (upper row) with mean frequencies ± SEM.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.


Figure 7A . Mean EAE clinical scores ( + SEM ) . Data consist of ( A ) n = 10 WT , n = 4 IL - 1R1 - / - PBS , n = 4 WT , n = 4 IL - 1R1ΔT and n = 5 IL - 1R1 - / - PC61 treated mice . B . Maximal EAE clinical scores ( individual plots with mean ) of selected mice . C . Total cell numbers ( mean + SEM ) of infiltrates isolated from the CNS of mice , shown in ( B ) . D . Analysis of IL - 1R1 expression by Th17 cells isolated from CNS of EAE diseased mice , shown in ( B ) . Data are representative FACS histogram overlays of cells isolated from ( grey ) wild type mice treated with PBS , ( black ) wild type , ( blue ) IL - 1R1ΔT and ( red ) IL - 1R1 - / - mice treated with PC61 , gated on VD - TCRβ + CD4 + IL - 17A + cells . ( E ) Frequencies of Th17 cells positive for IL - 1R1 , shown in ( D ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 7A. Mean EAE clinical scores (+SEM).Data consist of (A) n=10 WT, n=4 IL-1R1-/- PBS, n = 4 WT, n=4 IL-1R1ΔT and n=5 IL-1R1-/- PC61 treated mice.B. Maximal EAE clinical scores (individual plots with mean) of selected mice.C. Total cell numbers (mean +SEM) of infiltrates isolated from the CNS of mice, shown in (B).D. Analysis of IL-1R1 expression by Th17 cells isolated from CNS of EAE diseased mice, shown in (B). Data are representative FACS histogram overlays of cells isolated from (grey) wild type mice treated with PBS, (black) wild type, (blue) IL-1R1ΔT and (red) IL-1R1-/-mice treated with PC61, gated on VD-TCRβ+CD4+IL-17A+cells.(E) Frequencies of Th17 cells positive for IL-1R1, shown in (D).


Figure 1A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone ( HF ) for 5 h . B Quantification of ATF4 and P - eIF2α from experiments as in ( A ) . Data are mean ± SD ( n = 3 biological replicates ) . * p ≤ 0 . 0414 , * * p ≤ 0 . 0019 , * * * * p < 0 . 0001 , as determined by one - way ANOVA with Dunnett ' s multiple comparison test . Data information : Representative results of at least three independent experiments are shown in each panel . C Similar to ( A ) , but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel . D Similar to ( A ) , but using lysates from HeLa cells treated with 62 . 5 nM and 312 . 5 nM HF for indicated times . Data information : Representative results of at least three independent experiments are shown in each panel .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone (HF) for 5 h. B Quantification of ATF4 and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0414, **p ≤ 0.0019, ****p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparison test. Data information: Representative results of at least three independent experiments are shown in each panel.C Similar to (A), but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.D Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF for indicated times. Data information: Representative results of at least three independent experiments are shown in each panel.


Figure 2A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts ( MEFs ) of indicated genotype after treatment with 12 . 5 nM or 200 nM HF for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel . B Similar to ( A ) , but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h . C Similar to ( A ) , but with high HF concentrations . Data information : Representative results of at least three independent experiments are shown in each panel .

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Figure 2A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.B Similar to (A), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h. C Similar to (A), but with high HF concentrations. Data information: Representative results of at least three independent experiments are shown in each panel.


Figure 4B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF . C Similar to ( B ) , but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF . Data information : Representative results of at least three independent experiments are shown in each panel . D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8 - activating enzyme inhibitor MLN4924 ( 1 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel . E . Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG - 132 ( 10 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel .

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Figure 4B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF. C Similar to (B), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF. Data information: Representative results of at least three independent experiments are shown in each panel.D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924 (1 μM). Data information: Representative results of at least three independent experiments are shown in each panel.E. Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG-132 (10 μM). Data information: Representative results of at least three independent experiments are shown in each panel.


Figure 6A Newly synthesized proteins pulse - labeled with 35S - methionine for 10 min in HeLa cells pre - treated with indicated compounds for 2 . 5h , except ( E ) ( Bottom ) Coomassie - stained gel . B Newly synthesized proteins in HeLa cells treated with 2 . 5 μg / ml Tm or 50 μg / ml cycloheximide ( CHX ) . C Newly synthesized proteins in 4KO or 4KO + GCN2 MEF cells treated with 12 . 5 or 200 nM HF . D Newly synthesized proteins in eIF2αS / S and eIF2αA / A MEF cells treated with 12 . 5 or 200 nM HF . E Newly synthesized proteins in eIF2αS / S and eIF2αA / A MEF cells treated with indicated concentrations of Tm for 2 . 5 or 5 h . F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF . G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation ( 10 mM ) . H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation ( 10 mM ) . I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol ( His ) or borrelidin ( Bor ) .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Newly synthesized proteins pulse-labeled with 35S-methionine for 10 min in HeLa cells pre-treated with indicated compounds for 2.5h, except (E) (Bottom) Coomassie-stained gel.B Newly synthesized proteins in HeLa cells treated with 2.5 μg/ml Tm or 50 μg/ml cycloheximide (CHX).C Newly synthesized proteins in 4KO or 4KO+GCN2 MEF cells treated with 12.5 or 200 nM HF.D Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with 12.5 or 200 nM HF.E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM).H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM).I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin (Bor).


Figure 7G , H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 ( G ) and H23 ( H ) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose - response ( IC50 ) . Each point represents mean ( n = 3 biological replicates ) ± SD .

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Figure 7G, H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 (G) and H23 (H) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose-response (IC50). Each point represents mean (n = 3 biological replicates) ± SD.


Figure 1 . ( A ) HMGB1 translocates from the nucleus to the cytosol during autophagy , but not apoptosis . Mouse embryonic fibroblasts ( MEFs ) and the human Panc2 . 03 tumor cell line were treated with 1 µM rapamycin ( Rap ) for 12 h , starvation ( HBSS ) for 3 h , or UV irradiation at 50 mJ / cm2 for 5 min before a 12 - h recovery and then were immunostained with HMGB1 - specific antibody ( green ) and Hoechst 33342 ( blue ) . The mean nuclear ( Nuc ) and cytosolic ( Cyt ) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods . * , P < 0 . 05 and * * , P < 0 . 005 versus untreated ( UT ) group ; n = 3 . Representative images are depicted ( right ) . ( B ) Inhibition of autophagy blocks HMGB1 translocation . Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5 - specific shRNA for 48 h and were stimulated with starvation ( HBSS ) for 3 h and immunostained with HMGB1 - or LC3 - specific antibody and Hoechst 33342 . The mean nuclear / cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis . A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted ( right ) . In parallel , the indicated cells were transfected with GFP - LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP - LC3 punctae . * , P < 0 . 05 , * * , P < 0 . 005 , and * * * , P < 0 . 0005 versus HBSS group ; n = 3 . Ctrl , control . ( C ) Knockdown of ATG5 inhibits LC3 - II expression . Western blot analysis of LC3 - I / II expression in Panc02 cells under the conditions described in B . Actin was used as a loading control . ( D ) Hmgb1 − / − does not influence ATG5 staining . Hmgb1 − / − and Hmgb1 + / + MEFs were immunostained with HMGB1 - specific antibody ( green ) , ATG - 5 - specific antibody ( red ) , and Hoechst 33342 ( dark blue ) . Representative images are depicted in the right panels . ( E ) mETC inhibitors promote ROS production , autophagy , and HMGB1 translocation . Panc2 . 03 cells were stimulated with rotenone ( Rot ) , thenoyltrifluoroacetone ( TTFA ) , and antimycin A ( AA ) at indicated doses for 12 h , and ROS production was assessed by measuring the fluorescent intensity of CM - H2DCFDA in a fluorescent plate reader . In parallel experiments , cells were then immunostained with HMGB1 - or LC3 - specific antibody and Hoechst 33342 . The mean nuclear / cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis . * , P < 0 . 05 , * * , P < 0 . 005 , and * * * , P < 0 . 0005 versus untreated group ; n = 3 . ( F ) mETC inhibitors increase LC3 - II expression and promote HMGB1 translocation . Western blot analysis of LC3 - I / II and nuclear / cytosolic HMGB1 expression as indicated in E . Fibrillarin is a nuclear fraction control , and tubulin is a cytoplasmic fraction control . ( G ) Antioxidant and SOD RNAi limit starvation - induced autophagy and HMGB1 translocation . Panc2 . 03 cells were pretreated with the antioxidant ( NAC ) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h . Then cells were starved ( HBSS ) for 3 h and analyzed by imaging cytometry to determine the mean nuclear / cytosolic HMGB1 intensity and LC3 punctae per cell . * , P < 0 . 05 ; * * , P < 0 . 005 ; and * * * , P < 0 . 0005 versus HBSS group ; n = 3 . A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here . ( H ) Antioxidant and SOD RNAi limit starvation - induced autophagy as measured by LC3 - II expression . Western blot analysis of LC3 - I / II expression under the conditions indicated in G . Actin was used as a loading control . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(A) HMGB1 translocates from the nucleus to the cytosol during autophagy, but not apoptosis. Mouse embryonic fibroblasts (MEFs) and the human Panc2.03 tumor cell line were treated with 1 µM rapamycin (Rap) for 12 h, starvation (HBSS) for 3 h, or UV irradiation at 50 mJ/cm2 for 5 min before a 12-h recovery and then were immunostained with HMGB1-specific antibody (green) and Hoechst 33342 (blue). The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods. *, P < 0.05 and **, P < 0.005 versus untreated (UT) group; n = 3. Representative images are depicted (right).(B) Inhibition of autophagy blocks HMGB1 translocation. Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5-specific shRNA for 48 h and were stimulated with starvation (HBSS) for 3 h and immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted (right). In parallel, the indicated cells were transfected with GFP-LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP-LC3 punctae. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus HBSS group; n = 3. Ctrl, control.(C) Knockdown of ATG5 inhibits LC3-II expression. Western blot analysis of LC3-I/II expression in Panc02 cells under the conditions described in B. Actin was used as a loading control.(D) Hmgb1−/− does not influence ATG5 staining. Hmgb1−/− and Hmgb1+/+ MEFs were immunostained with HMGB1-specific antibody (green), ATG-5-specific antibody (red), and Hoechst 33342 (dark blue). Representative images are depicted in the right panels.(E) mETC inhibitors promote ROS production, autophagy, and HMGB1 translocation. Panc2.03 cells were stimulated with rotenone (Rot), thenoyltrifluoroacetone (TTFA), and antimycin A (AA) at indicated doses for 12 h, and ROS production was assessed by measuring the fluorescent intensity of CM-H2DCFDA in a fluorescent plate reader. In parallel experiments, cells were then immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus untreated group; n = 3.(F) mETC inhibitors increase LC3-II expression and promote HMGB1 translocation. Western blot analysis of LC3-I/II and nuclear/cytosolic HMGB1 expression as indicated in E. Fibrillarin is a nuclear fraction control, and tubulin is a cytoplasmic fraction control.(G) Antioxidant and SOD RNAi limit starvation-induced autophagy and HMGB1 translocation. Panc2.03 cells were pretreated with the antioxidant (NAC) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h. Then cells were starved (HBSS) for 3 h and analyzed by imaging cytometry to determine the mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus HBSS group; n = 3. A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here.(H) Antioxidant and SOD RNAi limit starvation-induced autophagy as measured by LC3-II expression. Western blot analysis of LC3-I/II expression under the conditions indicated in G. Actin was used as a loading control. Data are means ± SEM.


Figure 2 . ( A ) HMGB1 knockout inhibits LC3 punctae formation . HMGB1 − / − and HMGB1 + / + MEFs were treated with autophagic stimuli as indicated , and LC3 punctae formation was detected by LC3 antibody or GFP - LC3 as described in Materials and methods . * , P < 0 . 05 ; * * , P < 0 . 005 ; and * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . UT , untreated . ( B ) Representative images of LC3 punctae in Hmgb1 − / − and HMGB1 + / + MEFs with the indicated treatments are depicted . The percentage of cells showing accumulation of LC3 punctae was reported in A . ( C ) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A ( pepA ) at 10 µg / ml and E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 05 versus Hmgb1 + / + group ; n = 3 . Actin was used as a loading control . AU , arbitrary units . ( D ) Up - regulation of HMGB1 protein expression restores starvation - induced autophagy . Hmgb1 − / − MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h . LC3 punctae formation was assayed by imaging cytometric analysis . * * , P < 0 . 05 versus vector group ; n = 3 . Non , nontransfected . ( E ) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg / ml and E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 005 and * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . Representative images are depicted ( left ) . ( F ) Ultrastructural features in Hmgb1 − / − and Hmgb1 + / + MEFs with or without starvation ( HBSS for 3 h ) treatment ( a - e point to autophagosomes and autolysosomes ) . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(A) HMGB1 knockout inhibits LC3 punctae formation. HMGB1−/− and HMGB1+/+ MEFs were treated with autophagic stimuli as indicated, and LC3 punctae formation was detected by LC3 antibody or GFP-LC3 as described in Materials and methods. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. UT, untreated. (B) Representative images of LC3 punctae in Hmgb1−/− and HMGB1+/+ MEFs with the indicated treatments are depicted. The percentage of cells showing accumulation of LC3 punctae was reported in A.(C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units.(D) Up-regulation of HMGB1 protein expression restores starvation-induced autophagy. Hmgb1−/− MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h. LC3 punctae formation was assayed by imaging cytometric analysis. **, P < 0.05 versus vector group; n = 3. Non, nontransfected.(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. Representative images are depicted (left).(F) Ultrastructural features in Hmgb1−/− and Hmgb1+/+ MEFs with or without starvation (HBSS for 3 h) treatment (a-e point to autophagosomes and autolysosomes). Data are means ± SEM.


Figure 3 . Inhibition of autophagy by cytoplasmic HMGB1 . Hmgb1 − / − and Hmgb1 + / + MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h , and LC3 punctae formation was detected by a confocal microscope . ( A ) Representative images of LC3 punctae ( white arrows ) and HMGB1 ( red arrows ) in cytoplasts of Hmgb1 − / − and Hmgb1 + / + MEFs are depicted . ( B ) The percentage of cells showing accumulation of LC3 punctae was reported ( * , P < 0 . 05 ; n = 3 ) . Data are means ± SEM .

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Figure 3.Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.


Figure 4 . ( A ) MEK inhibitors block starvation - induced p - ERK and Bcl - 2 . HMGB1 − / − and HMGB1 + / + MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h . Protein expression levels were assessed as indicated by co - IP or Western blotting . ( B ) Knockout of HMGB1 limits the disassociation of the Bcl - 2 - Beclin1 complex during treatment with autophagic stimuli . HMGB1 − / − and HMGB1 + / + MEFs were starved in the presence or absence of 10 µM U0126 for 3 h . Protein expression levels were then assayed as indicated by co - IP or Western blotting . ( C ) HMGB1 interacts directly with Beclin1 during autophagy . HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co - IP or Western blotting . ( D ) HMGB1 direct interactions with Bcl - 2 are dependent on Beclin1 . Knockdown of Beclin1 and Bcl - 2 by siRNA in HMGB1 wild - type MEFs was performed , and protein expression levels were then assayed as indicated by co - IP or Western blotting . Ctrl , control . ( E and F ) Quantitative data demonstrating the interaction between HMGB1 - Beclin1 and Beclin1 - HMGB1 using densitometry software to assay the relative protein band intensity in co - IP experiments as shown in A - D . * * , P < 0 . 005 and * * * , P < 0 . 0005 ; n = 3 . UT , untreated . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting.(B) Knockout of HMGB1 limits the disassociation of the Bcl-2-Beclin1 complex during treatment with autophagic stimuli. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting.(C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co-IP or Western blotting.(D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1 and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl, control.(E and F) Quantitative data demonstrating the interaction between HMGB1-Beclin1 and Beclin1-HMGB1 using densitometry software to assay the relative protein band intensity in co-IP experiments as shown in A-D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.