A ) siRNA screen performed in Caco - 2 cysts treated with Y27632 : Caco - 2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days . Then , Caco - 2 cysts were treated with Y27632 and the serum gradient was induced . After 2 days , the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining . The bar graph represents the mean invasion rate . Each condition was duplicated . The red dashed line represent the median invasion rate , the red full line indicates the 3 fold decrease in invasion rate . Three hits were identified : # 2 : ARHGEF18 + RNEF , # 7 : SGEF + ARHGEF16 and # 24 : FARP1 + FARP2 ( Mean ± SD ) .

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A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .


B ) Caco - 2 cysts stably expressing empty vector ( pGIPZ ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632 , allowed to invade for 2 days , fixed and stained for F - actin ( Phalloidin ) . Boxed regions i , ii , iii and iv are shown at high magnification . Arrowheads point to non - protruding cells , arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . C ) Bar graphs representing the percentage of protrusive cysts formed from Caco - 2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments ( Means ± SEM , paired t - test , ns : non significant , * * p < 0 . 01 ) .

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B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01).


D ) Representative acquisition of Caco - 2 cells overexpressing GFP - FARP2 grown in 2D and stained with ZO - 1 , in non - treated ( NT ) and after 2h with Y27632 or H1152 . The dashed lines crossing the cell - cell junctions ( i , ii and iii ) are representative of the linescans measurements . Scale bars : 20μm . E ) Representative fluorescence intensity of ZO - 1 ( red ) staining and GFP - FARP2 ( green ) along the dashed line i , ii and iii represented corresponding to non - treated , Y27632 or H1152 treatments )

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D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments)


F ) Bar graph representing the average ratio of GFP - FARP2 over ZO - 1 fluorescence intensities measured with linescans crossing cell - cell junctions . The quantification was performed on 71 cells expressing GFP - FARP2 - FL from 3 independent experiments . For each cell , between 10 and 15 lines ( 12 pixels wide ) were drawn across random region of cell - cell junctions positive for ZO - 1 staining ( Mean ± SEM Unpaired t - test * * p < 0 . 01 , * p < 0 . 05 ) .

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F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).


A ) Caco - 2 cysts stably expressing GFP ( treated or not with Y27632 ) , or Cherry - FARP2 - full length ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH 730 / 733 ( FARP2 - del / Mut ) were allowed to invade for 2 days , fixed and stained for F - actin . Boxed regions i , ii , iii , iv and v are shown at high magnification . Arrowheads point to non - protruding cells , arrow points to protruding cells , white star shows nucleus that engages in protrusion . Scale bars : 20μm . B ) Bargraph representing the percentage of protrusive cysts expressing GFP ( treated or not with Y27632 ) or the mentioned GFP - FARP2 constructs from 3 independent experiments ( Means ± SEM , paired t - test , * * p < 0 , 01 , n . s : non significant ) .

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A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant).


C ) Representative images of Caco - 2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin - IIA and allowed to invade for 2 days . After fixation , the cysts were stained for F - actin ( Phalloidin ) and nuclei ( DAPI ) . Representative Y27632 - induced « invasive » phenotype and Blebbistatin - induced « dendritic » phenotype are displayed . Arrows point to protruding cells , white star shows nucleus that engages in protrusion and arrowheads point to " dendritic " protrusions . D ) Bar graph representing the percentage of cysts with the " invasive " or " dendritic " phenotypes after treatment with Y27632 , Blebbistatin or siMyosin - IIA or siMyosin - IIB from at least 3 independent experiments ( Means ± SEM , unpaired t - test , * p < 0 , 05 , n . s : non significant ) .

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C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions. D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant).


E ) Mature Caco - 2 cysts were transduced with GFP or Cherry tagged FARP2 - full length ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH / 730 / 733 ( FARP2 - del / Mut ) , and treated ( or not , NT ) with Blebbistatin . 2 days after invasion , the cysts were fixed and stained for F - actin . Boxed regions i , ii , iii and iv are shown at high magnification . Arrowheads point to non - protruding cells ; arrow points to protruding cells and white stars show nucleus that engage in protrusions . Scale bars : 20 μm . F ) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin , from at least 3 independent experiments ( Means ± SEM , t - test , * * p < 0 , 01 , n . s : non significant ) .

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E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).


G IP of UbcH7 by PknG or PknGΔUbl in U937 cells . Cells were infected with WT , ΔpknG , or ΔpknG : pknGΔUbl Mtb strains as in B . H IP of UbcH7 by PknG or PknG E190A in U937 cells . Cells were infected with WT , ΔpknG , or ΔpknG : pknG E190A Mtb strains as in B .

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G IP of UbcH7 by PknG or PknGΔUbl in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknGΔUbl Mtb strains as in B. H IP of UbcH7 by PknG or PknG E190A in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknG E190A Mtb strains as in B.


E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His - UBA1 at 37 ° C for 30 min . The reaction products were analyzed by immunoblotting with antibodies against Ub ( top panel ) , the His - tag on UBA1 ( middle panel ) , and PknG ( bottom panel ) , or by staining with Coomassie Brilliant Blue ( bottom ) . For reducing conditions , samples were treated with 500 mM β - mercaptoethanol ( βME ) prior to SDS - PAGE gel analysis .

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E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol (βME) prior to SDS-PAGE gel analysis.


A MS / MS analysis of UbcH7 in the absence ( top ) or presence ( bottom ) of PknG to reveal ubiquitination sites . Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high - confidence peptides possessing di - Gly modifications . The ubiquitinated residue ( Lys82 ) in the peptide sequence is highlighted in red . The peak heights are the relative abundances of the corresponding fragmentation ions . Matched amino terminus - containing ions ( b ions ) are indicated in red , and matched carboxyl terminus - containing ions ( y ions ) are indicated in blue . Only the major peaks that were identified are labeled .

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A MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled.


A Luciferase assay of NF - κB activation in HEK293T cells transfected with WT or mutant Mtb PknG . The graph shows the mean ± SEM of three independent biological replicates , n = 3 . * P < 0 . 05 , * * P < 0 . 01 . Data were analyzed according to one - way analysis of variance ( ANOVA ) using GraphPad Prism 7 .

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A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7.


D Survival of Mtb strains in primary human monocyte - derived macrophages treated as in B and C . The graph shows the mean ± SEM of three independent biological replicates , n = 3 . * P < 0 . 05 , * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

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D Survival of Mtb strains in primary human monocyte-derived macrophages treated as in B and C. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.


B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells . Cells were infected with the indicated Mtb strains at an MOI of 1 . Non - infected cells were used as controls . After 4 hours , the cells were lysed and immunoprecipitated with the antibody against PknG . The immunoprecipitated proteins were immunoblotted with TRAF2 , TAK1 , TAB2 ( negative control ) , or TAB3 ( negative control ) antibodies .

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B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies.


E Immunoblotting of TRAF2 , TAK1 , and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours . Cells were treated with CHX ( 50 μg / mL ) with or without MG132 ( 5 μM ) . Cell lysates were analyzed by immunoblotting for TRAF2 ( anti - TRAF2 ) , TAK1 ( anti - TAK1 ) , and PknG ( anti - PknG ) . GAPDH was used as the loading control . Densitometric quantification of immunoblots is indicated below the immunoblots .

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E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.


G , H In vivo ubiquitination assay of TRAF2 ( G ) and TAK1 ( H ) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours . Cells were lysed and immunoprecipitated using the antibody against Flag . Immunoprecipitated proteins were immunoblotted with the antibody against HA .

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G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.


A , B qPCR analysis of Tnf mRNA ( A ) and Il6 mRNA ( B ) in splenic cells from C57BL / 6 mice infected with WT or mutant Mtb stains for 0 - 20 days . Data are representative of one experiment with two independent biological replicates ( mean ± SEM of n = 6 mice per group ) . * P < 0 . 05 and * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

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A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.


D H & E staining of lungs and livers of C57BL / 6 mice treated as in A . Arrows indicate puncta of cellular infiltration . Scale bars , 200 μm .

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D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm.


B , HeLa cells were treated with DMSO or 2µM Mito - C for 15 min and immunostained with anti - TOM20 ( green ) antibody and DAPI ; cropped areas show the mitochondria morphology changes .

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B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.


C , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in B ) from HeLa cells treated with DMSO or 2µM Mito - C for 15 min or 2h , expressed as ratio of fragmented mitochondria ( percentage of total ) . Errors bars show the standard error of the mean ( SEM ) ( n = 100 , technical replicates ) .

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C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).


E , Time lapse video - microscopy snapshot on HeLa cells transfected with NAF - 1 - mRFP ( red ) and treated with fluoMito - C ( green ) . The distance / intensity fluorescence quantification graph illustrates the codistribution of fluoMito - C and NAF - 1 - mRFP .

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E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.


A , Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA ; Bar Chart ( right panel ) shows replicates quantification of MitoNEET expression . Errors bars show the standard deviation ( SD ) of 3 independent experiments .

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A, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression. Errors bars show the standard deviation (SD) of 3 independent experiments.


B , Western blot analysis of NAF - 1 expression in HeLa cells transfected with CISD2 siRNA ; graph ( right panel ) shows the replicates quantification of NAF - 1 expression . Errors bars show the standard deviation ( SD ) of 3 independent experiments .

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B, Western blot analysis of NAF-1 expression in HeLa cells transfected with CISD2 siRNA; graph (right panel) shows the replicates quantification of NAF-1 expression. Errors bars show the standard deviation (SD) of 3 independent experiments.


D , HeLa cells transfected with siRNA to reduce expression of MitoNEET ( CISD1 ) , NAF - 1 ( CISD2 ) or MiNT ( CISD3 ) and immunostained with anti TOM20 antibody . Arrowheads indicate fragmented mitochondria .

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D, HeLa cells transfected with siRNA to reduce expression of MitoNEET (CISD1), NAF-1 (CISD2) or MiNT (CISD3) and immunostained with anti TOM20 antibody. Arrowheads indicate fragmented mitochondria.


E , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in D ) from HeLa cells transfected with siRNA targeting mitoNEET ( siCISD1 ) , NAF1 ( siCISD2 ) or MiNT ( siCISD3 ) . Quantification is expressed as ratio of tubular and fragmented mitochondria . Errors bars show the standard error of the mean ( SEM ) . ( n = 125 - 130 cells ) .

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E, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in D) from HeLa cells transfected with siRNA targeting mitoNEET (siCISD1), NAF1 (siCISD2) or MiNT (siCISD3). Quantification is expressed as ratio of tubular and fragmented mitochondria. Errors bars show the standard error of the mean (SEM). (n =125-130 cells).


F , Electron microscopy pictures from 150 INS - 1 cells transfected as described in G .

F, Electron microscopy pictures from 150 INS-1 cells transfected as described in G.


G , Western blot analysis of NAF - 1 expression in cells stably transfected with control shRNA , shRNA targeting reduction of NAF - 1 protein expression ( shCISD2 ) , or shRNA targeting reduction of NAF - 1 protein expression complemented with plasmid derived expression of NAF - 1 ( shCISD2 + CISD2 ) ; western blot quantification of NAF - 1 is shown on the accompanying bar chart . Errors bars show the standard deviation ( SD ) of 3 independent experiments .

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G, Western blot analysis of NAF-1 expression in cells stably transfected with control shRNA, shRNA targeting reduction of NAF-1 protein expression (shCISD2), or shRNA targeting reduction of NAF-1 protein expression complemented with plasmid derived expression of NAF-1 (shCISD2+CISD2); western blot quantification of NAF-1 is shown on the accompanying bar chart. Errors bars show the standard deviation (SD) of 3 independent experiments.


H , Quantification of mitochondrial length in EM images from cells transfected as described in G . Errors bars show the standard error of the mean ( SEM ) ( n = 260 - 270 , technical replicates ) .

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H, Quantification of mitochondrial length in EM images from cells transfected as described in G. Errors bars show the standard error of the mean (SEM) (n=260-270, technical replicates).


A , HeLa cells treated or not , with 2µM Mito - C for 15 min were immunostained with anti - TOM20 ( red ) and anti - DRP1 ( green ) ; arrowheads in the far - right panel indicate recruitment of DRP1 onto the mitochondrial surface ( TOM20 ) . B , Quantification of DRP1 signal on TOM20 positive structures . Errors bars show the standard error of the mean ( SEM ) ( n = 45 , technical replicates ) .

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A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). C


C , Western blot analysis of DRP1 , GAPDH ( cytosolic marker ) and TOM20 ( mitochondrial marker ) protein in cytosolic and mitochondrial fractions treated or not , with 2µM Mito - C as indicated . D , Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions . Errors bars show the standard deviation ( SD ) of 5 independent experiments .

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C, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated. D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments. E


E , HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not , with Mito - C ( T for transfected , NT for not transfected ) . F , Quantification of the mitochondrial phenotypes observed in E . Errors bars show the standard error of the mean ( SEM ) . ( 30 - 35 images each with an average of 15 - 20 cells from triplicate independent experiments were analyzed ) .

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E, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected). F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed). G


G , Electron micrographs ( EM ) from HeLa cells treated with 2µM Mito - C or DMSO for 120 minutes . Empty arrowheads indicate ER - mitochondria contact sites . H , Quantification of ER - mitochondria membrane contact sites events ( reported in 50µm² section ; 50 images from an average of 30 cells from triplicate independent experiments were analyzed ) . Errors bars show the standard error of the mean ( SEM ) . I , Quantification of ER - mitochondria membrane contact sites density ( in µm ; 30 images from an average of 30 cells from triplicate independent experiments were analyzed ) . Errors bars show the standard error of the mean ( SEM ) .

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G, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites. H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). Da


B , Huh7 cells were treated with or without ( DMSO ) Mito - C , infected with dengue virus and fixed after 48h of infection . Cells were then immunostained for endogenous TOM20 ( green channel ) , viral non - structural protein NS5 ( red ) and DAPI ( Blue ) . Cropped areas illustrate the mitochondrial morphology in described conditions .

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B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions.


C , Quantification of the mitochondrial phenotypes observed in b . Errors bars show the standard error of the mean ( SEM ) . ( 15 - 20 images each with an average of 20 - 25 cells from triplicate independent experiments were analyzed ) .

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C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).


A - C . Enzyme - linked immunosorbent assay ( ELISA ) of IL - 6 , TNF - α and IFN - β protein and real - time PCR analysis of IL - 6 , TNF - α and IFN - β mRNA in peritoneal macrophage isolated from wild type and RKIP - deficient mice treated with VSV ( MOI , 0 . 1 ) ( A ) .

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A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with VSV (MOI,0.1) (A).


A - C . Enzyme - linked immunosorbent assay ( ELISA ) of IL - 6 , TNF - α and IFN - β protein and real - time PCR analysis of IL - 6 , TNF - α and IFN - β mRNA in peritoneal macrophage isolated from wild type and RKIP - deficient mice treated with SEV ( MOI , 10 ) ( B ) .

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A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with SEV (MOI,10) (B).


A - C . Enzyme - linked immunosorbent assay ( ELISA ) of IL - 6 , TNF - α and IFN - β protein and real - time PCR analysis of IL - 6 , TNF - α and IFN - β mRNA in peritoneal macrophage isolated from wild type and RKIP - deficient mice transfected with poly ( I : C ) ( 1 μg / ml ) ( C ) .

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A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice transfected with poly(I:C) ( 1 μg/ml) (C).


D . Fluorescence - activated cell sorting ( FACS ) and TCID50 ( 50 % tissue culture infective dose ) assessing the proliferation level of VSV . RAW264 . 7 cells line stably overexpressed flag - RKIP was infected with VSV - eGFP at MOI of 1 for the indicated time . The fusion protein fluorophore were analyzed by FACS . Then fresh medium with the VSV - eGFP infection was serially diluted on the monolayer of HEK293 cells for 3 - 7 days and TCID50 was measured .

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D. Fluorescence-activated cell sorting (FACS) and TCID50 (50% tissue culture infective dose) assessing the proliferation level of VSV. RAW264.7 cells line stably overexpressed flag-RKIP was infected with VSV-eGFP at MOI of 1 for the indicated time. The fusion protein fluorophore were analyzed by FACS. Then fresh medium with the VSV-eGFP infection was serially diluted on the monolayer of HEK293 cells for 3-7 days and TCID50 was measured.


E . FACS analysis the infection of wild type and RKIP - deficient peritoneal macrophage infected with VSV - eGFP at MOI of 1 . The fresh medium with the VSV - eGFP infection 9 hr were collected for TCID50 assessing .

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E. FACS analysis the infection of wild type and RKIP-deficient peritoneal macrophage infected with VSV-eGFP at MOI of 1. The fresh medium with the VSV-eGFP infection 9 hr were collected for TCID50 assessing.


A . Wide - type ( RKIP + / + ) and RKIP deficiency ( RKIP - / - ) chimera were intraperitoneally injected with VSV ( 1x107 pfu / g ) , 12 hours later , ELISA of IFN - β and IL - 6 in serum ( n = 6 per group ) .

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A. Wide-type (RKIP+/+) and RKIP deficiency (RKIP-/-) chimera were intraperitoneally injected with VSV (1x107 pfu/g), 12 hours later, ELISA of IFN-β and IL-6 in serum (n=6 per group).


B . Determination of VSV loads in organs by TCID50 assay 12 hours later after RKIP + / + and RKIP - / - chimera mice ( n = 6 per group ) intraperitoneally injected with VSV ( 1x107 pfu / g ) .

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B. Determination of VSV loads in organs by TCID50 assay 12 hours later after RKIP+/+ and RKIP-/- chimera mice (n = 6 per group) intraperitoneally injected with VSV (1x107 pfu/g).


C . Real - time PCR analysis of IFN - β mRNA and VSV expression in liver from RKIP + / + and RKIP - / - chimera mice 12 hours later intraperitoneally injected with VSV ( 1x107 pfu / g ) ( n = 6 per group ) .

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C. Real-time PCR analysis of IFN-β mRNA and VSV expression in liver from RKIP+/+ and RKIP-/- chimera mice 12 hours later intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).


D . Pathology of wild type and RKIP deficient chimeric mice in response to VSV infection . Hematoxylin and eosin staining of liver and lung sections from mice in ( A ) . Scale bar , 100 μm ( left ) or 50 μm ( right ) .

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D. Pathology of wild type and RKIP deficient chimeric mice in response to VSV infection. Hematoxylin and eosin staining of liver and lung sections from mice in (A). Scale bar, 100 μm (left) or 50 μm (right).


E . Real - time PCR analysis of IFN - β mRNA expression in peritoneal macrophages from RKIP + / + and RKIP - / - chimera mice 12 hours after intraperitoneally injected with VSV ( 1x107 pfu / g ) ( n = 6 per group ) .

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E. Real-time PCR analysis of IFN-β mRNA expression in peritoneal macrophages from RKIP+/+ and RKIP-/- chimera mice 12 hours after intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).


F . Survival curve for 8 weeks - old wild type and RKIP deficient chimeras challenged with VSV ( 1x108 pfu / g , i . v . ) ( n = 10 per group ) . * p < 0 . 05 Wilcoxon Test .

F. Survival curve for 8 weeks-old wild type and RKIP deficient chimeras challenged with VSV (1x108 pfu/g, i.v.) (n=10 per group). *p<0.05 Wilcoxon Test .


A . Immunoblot analysis of p - TBK1 , - IKKi , - IRF3 , - P38 , - JNK1 / 2 , - ERK1 / 2 and - IκBα in lysates of RKIP + / + , RKIP + / - and RKIP - / - peritoneal macrophages stimulated with VSV ( MOI , 0 . 1 ) for the indicated time .

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A. Immunoblot analysis of p-TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophages stimulated with VSV (MOI, 0.1) for the indicated time.


B . Immunoblot assay of RKIP + / + , RKIP + / - and RKIP - / - peritoneal macrophage cells treated with intracellular poly ( I : C ) ( 1 μg / ml ) .

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B. Immunoblot assay of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophage cells treated with intracellular poly(I:C) (1 μg/ml).


C . Immunoblot analysis of phosphorylated ( p - ) TBK1 , IKKi , IRF3 , P38 , JNK1 / 2 , ERK1 / 2 in peritoneal macrophage transfected with RKIP - specific or scrambled siRNA , followed by infection with VSV ( MOI , 0 . 1 ) . β - actin was used as a loading control .

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C. Immunoblot analysis of phosphorylated (p-) TBK1, IKKi, IRF3, P38, JNK1/2, ERK1/2 in peritoneal macrophage transfected with RKIP-specific or scrambled siRNA, followed by infection with VSV (MOI, 0.1). β-actin was used as a loading control.


D . Immunoblot analysis of p - TBK1 , - IKKi , - IRF3 , - P38 , - JNK1 / 2 , - ERK1 / 2 and - IκBα in lysates of VSV - treated RAW264 . 7 cells stably over - expressing RKIP .

D. Immunoblot analysis of p- TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of VSV-treated RAW264.7 cells stably over-expressing RKIP.


E . Immunoblot analysis in lysates of RAW264 . 7 cells stably overexpressing RKIP treated with intracellular poly ( I : C ) ( 1 μg / ml ) .

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E. Immunoblot analysis in lysates of RAW264.7 cells stably overexpressing RKIP treated with intracellular poly(I:C) (1 μg/ml).


F . Luciferase activity in 293T cells transfected with plasmid encoding a luciferase reporter for IFN - β ( IFN - β - luc ) or ISRE ( ISRE - luc ) , and an expression vector for RKIP ( 0 , 50 , 100 , 200 ng each ) , together with plasmids encoding RIG - I CARD , TBK1 , IKKi and IRF3 5D .

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F. Luciferase activity in 293T cells transfected with plasmid encoding a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc), and an expression vector for RKIP (0, 50, 100, 200 ng each), together with plasmids encoding RIG-I CARD, TBK1, IKKi and IRF3 5D.


A . Immunoblot of extracts of RAW264 . 7 cells stably overexpressing flag - RKIP infected with VSV ( MOI , 0 . 1 ) for the indicated times followed by immunoprecipitation ( IP ) . WCL , whole cell lysates .

A. Immunoblot of extracts of RAW264.7 cells stably overexpressing flag-RKIP infected with VSV (MOI, 0.1) for the indicated times followed by immunoprecipitation (IP). WCL, whole cell lysates.


B . Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc - RKIP alone or with the deletion mutants of TBK1 . Numbers in parentheses indicate amino acids included in construct . FL , full length ; KD , kinase domain ; ULD , ubiquitin - like domain ; SDD , α - helical scaffold domain ; NBD , C - terminal domain . * indicates targeted band .

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B. Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc-RKIP alone or with the deletion mutants of TBK1. Numbers in parentheses indicate amino acids included in construct. FL, full length; KD, kinase domain; ULD, ubiquitin-like domain; SDD, α-helical scaffold domain; NBD, C-terminal domain.* indicates targeted band.


C . Immunoblot of 293T cells transfected with vector for myc - TBK1 together with plasmid for HA - GSK3β or HA - RKIP , followed by immunoprecipitation . * indicates targeted band .

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C. Immunoblot of 293T cells transfected with vector for myc-TBK1 together with plasmid for HA-GSK3β or HA-RKIP, followed by immunoprecipitation. * indicates targeted band.


D . In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP , 100 μM GST - RKIP or 100 μM GST - GFP and 5 μM flag - TBK1 at 30 ° C for 30 min and then followed by immunoblotting with the indicated Antibody .

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D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.


A . Real - time PCR analysis of IL - 6 , TNF - α and IFN - β mRNA in RAW264 . 7 cells stably overexpressing flag - RKIP , flag - S109A and flag - S109D , followed by infection with VSV ( MOI , 0 . 1 ) .

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A. Real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, followed by infection with VSV (MOI, 0.1).


B . Immunoblot analysis of p - TBK1 , - IRF3 , - P38 of cells as in ( A ) .

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B. Immunoblot analysis of p- TBK1, -IRF3, -P38 of cells as in (A).


C . FACS analysis of RAW264 . 7 cells stably overexpressing flag - RKIP , flag - S109A and flag - S109D infected with VSV - eGFP at MOI of 1 .

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C. FACS analysis of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D infected with VSV-eGFP at MOI of 1.


D . Immunoblot analysis of phosphorylated - TBK1 , - IRF3 , - P38 in lysates of RKIP + / + or RKIP - / - bone marrow derived macrophages ( BMDM ) transfected with flag - RKIP , flag - S109A and flag - S109D , and then stimulated with VSV ( MOI , 0 . 1 ) .

D. Immunoblot analysis of phosphorylated-TBK1, -IRF3, -P38 in lysates of RKIP+/+ or RKIP-/- bone marrow derived macrophages (BMDM) transfected with flag-RKIP, flag-S109A and flag-S109D, and then stimulated with VSV (MOI, 0.1).


E . ELISA of IFN - β and TNF - α of cells in ( D ) .

E. ELISA of IFN-β and TNF-α of cells in (D).


A . BMDM cells were infected with VSV ( MOI , 0 . 1 ) or HSV ( MOI , 5 ) for the indicated times . Cell lysates were analyzed by immunoblotting with the indicated antibodies .

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A. BMDM cells were infected with VSV (MOI, 0.1) or HSV (MOI, 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies.


B . BMDM cells were transfected with control or TBK1 siRNA . 72 hours after transfection , cells were analyzed by immunoblotting with antibody for phospho - RKIP or TBK1 .

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B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1.


C . Immunoblot analysis of RKIP phosphorylation in control or TBK1 - / - mouse embryonic fibroblasts after VSV ( MOI , 0 . 1 ) infection for the indicated times .

C. Immunoblot analysis of RKIP phosphorylation in control or TBK1-/- mouse embryonic fibroblasts after VSV (MOI, 0.1) infection for the indicated times.


D . Recombinant flag - tagged TBK1 was immunoprecipitated with anti - flag M2 beads in flag - TBK1 transfected 293T cells . Followed by in vitro kinase assay , which was carried out in the reaction mixture containing 2 mM ATP , 100 μM GST - RKIP or 100 μM GST - GFP together with 5 μM flag - TBK1 at 30 ° C for 30 min . Assay mixtures were immunoblotted with the indicated antibody for phospho - RKIP , flag , GST or his .

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D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his.


E . Immunoblot analysis of extracts of RAW264 . 7 cells stably overexpressing flag - RKIP , flag - S109A and flag - S109D , infected with VSV ( MOI , 0 . 1 ) for the indicated times , followed by immunoprecipitation with anti - flag beads .

E. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, infected with VSV (MOI, 0.1) for the indicated times, followed by immunoprecipitation with anti-flag beads.


F . BMDM cells were infected with VSV ( MOI , 0 . 1 ) for the indicated times . The cell lysates were immunoprecipitated with anti - TBK1 ( left ) or anti - RKIP ( right ) . The immunoprecipitates were analyzed by immunoblot with anti - RKIP or anti - TBK1 antibody . The levels of the endogenous RKIP , TBK1 , and p - RKIP were detected by immunoblot analysis .

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F. BMDM cells were infected with VSV (MOI, 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 (left) or anti-RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti-RKIP or anti-TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p-RKIP were detected by immunoblot analysis.


G . Co - immunoprecipitation and immunoblot analysis of 293T cells transfected with myc - TBK1 alone or with flag - RKIP , flag - S109A or flag - S109D .

G. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with myc-TBK1 alone or with flag-RKIP, flag-S109A or flag-S109D.


H . GST - pull down experiment was performed with 1 μg fusion protein GST - GFP , GST - his - RKIP or GST - his - S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium , followed by adding 1 μg flag - TBK1 and incubating at 4 ° C for 3 hours with gentle rotation . Pull down ( 1st - 3th lane ) and input samples ( 4th lane ) were separated by SDS - PAGE followed by immunoblotting with anti - GST or anti - flag antibody .

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H. GST-pull down experiment was performed with 1 μg fusion protein GST-GFP, GST-his-RKIP or GST-his-S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag-TBK1 and incubating at 4°C for 3 hours with gentle rotation. Pull down (1st-3th lane) and input samples (4th lane) were separated by SDS-PAGE followed by immunoblotting with anti-GST or anti-flag antibody.


I . Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc - TBK1 , flag - RKIP , flag - S109A , flag - S109D and HA - GSK3β or flag - GFP .

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I. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc-TBK1, flag-RKIP, flag-S109A, flag-S109D and HA-GSK3β or flag-GFP.


J . In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP , 50 μM GST - TBK1 together with 100 μM GST - his - RKIP , 100 μM GST - his - S109D , 100 μM GST - his - S109A or 100 μM GST , respectively . React at 30 ° C for 30 min . Then followed by immunoblotting with anti - p - TBK1or anti - GST antibody .

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

J. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.


( A - B ) Human fibroblasts ( BJ ) were treated with vehicle ( water for 8 times 24 hours ) or abemaciclib ( 1 μM for 8 times 24 hours ) . The population doubling over the 8 days treatment is plotted ( A ) . 1 or 8 dpt , cells were incubated with EdU for 20 hours and stained and quantified ( n = 9 samples from 3 independent experiments ; scale bar , 150 μm ) ( B ) . Data information : Data are means ± SD . Unpaired student t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . dpt , days post treatment .

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

(A-B) Human fibroblasts (BJ) were treated with vehicle (water for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). The population doubling over the 8 days treatment is plotted (A). 1 or 8 dpt, cells were incubated with EdU for 20 hours and stained and quantified (n=9 samples from 3 independent experiments; scale bar, 150 μm) (B). Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001. dpt, days post treatment.


( C ) Chromatin was extracted from BJ cells treated with vehicle or abemaciclib ( 1 μM for 8 times 24 hours ) or nutlin - 3a ( 10 μM for 8 times 24 hours ) or doxorubicin ( 250 nM for 24 hours ) and ChIP assays using an antibody against p53 were performed . qRT - PCR was performed using primers amplifying promoter regions of CDKN1A , GADD45A and MDM2 genes containing p53 binding sites . Values indicate fold enrichment relative to the vehicle group ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) Chromatin was extracted from BJ cells treated with vehicle or abemaciclib (1 μM for 8 times 24 hours) or nutlin-3a (10 μM for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) and ChIP assays using an antibody against p53 were performed. qRT-PCR was performed using primers amplifying promoter regions of CDKN1A, GADD45A and MDM2 genes containing p53 binding sites. Values indicate fold enrichment relative to the vehicle group (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( E ) 3 * 103 BJ cells of the indicated genotypes were re - plated after vehicle or abemaciclib treatment ( 1 μM ) and stained with 0 . 2 % crystal violet 8 dpt ( n = 3 independent experiments ) . ( F ) 8 dpt , treated BJ cells were incubated with EdU for 20 hours and stained ( n = 9 samples from 3 independent experiments ) . ( Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) 3*103 BJ cells of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (1 μM) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (F) 8 dpt, treated BJ cells were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( G ) 3 * 103 mouse dermal fibroblasts ( MDFs ) of the indicated genotypes were re - plated after vehicle or abemaciclib treatment ( 4 μM for 8 times 24 hours ) and stained with 0 . 2 % crystal violet 8 dpt ( n = 3 independent experiments ) . ( H ) 8 dpt , treated MDFs were incubated with EdU for 20 hours and stained ( n = 9 samples from 3 independent experiments ) . ( Data information : Data are means ± SD . Unpaired student t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(G) 3*103 mouse dermal fibroblasts (MDFs) of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (4 μM for 8 times 24 hours) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (H) 8 dpt, treated MDFs were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001.


( I ) RNA was isolated from vehicle or abemaciclib ( 1 μM ) treated scramble / shp53 BJ cells and mRNA for the indicated genes quantified by qRT - PCR relative to tubulin ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(I) RNA was isolated from vehicle or abemaciclib (1 μM) treated scramble/shp53 BJ cells and mRNA for the indicated genes quantified by qRT-PCR relative to tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . n = 6 mice / group . 14 dpt , bioluminescence was visualized and quantified by the IVIS spectrum in vivo imaging system , as shown by representative bioluminescence images ( A ) and quantification ( B ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). n=6 mice/group. 14 dpt, bioluminescence was visualized and quantified by the IVIS spectrum in vivo imaging system, as shown by representative bioluminescence images (A) and quantification (B). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . n = 6 mice / group . RNA isolated from kidneys and mRNA encoding p16 quantified by qRT - PCR ( C ) . SA - β - gal activities in vehicle - , doxorubicin - or abemaciclib - treated mouse kidney sections at 15 dpt quantified ( E ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). n=6 mice/group. RNA isolated from kidneys and mRNA encoding p16 quantified by qRT-PCR (C). SA-β-gal activities in vehicle-, doxorubicin- or abemaciclib-treated mouse kidney sections at 15 dpt quantified (E). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


( G ) RNA was isolated from abemaciclib ± pifithrin - α treated kidneys and mRNA encoding p16 and p21 genes was quantified by qRT - PCR and normalized on tubulin ( n = 5 mice / group ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

(G) RNA was isolated from abemaciclib ± pifithrin-α treated kidneys and mRNA encoding p16 and p21 genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


For the doxorubicin or abemaciclib treated mice ( n = 6 mice / group ) , Percentage of T cells , B cells , granulocytes and macrophages were determined by flow cytometry analysis ( N ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

For the doxorubicin or abemaciclib treated mice (n=6 mice/group), Percentage of T cells, B cells, granulocytes and macrophages were determined by flow cytometry analysis (N). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


( A ) 8 dpt , cells were stained for γ - H2AX ( scale bar , 60 μm ) and quantified ( vehicle = 288 cells , doxorubicin = 280 cells , palbociclib = 276 cells , abemaciclib = 266 cells from 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant . Doxo , doxorubicin . Abe , abemaciclib . dpt , days post treatment .

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

(A) 8 dpt, cells were stained for γ-H2AX (scale bar, 60 μm) and quantified (vehicle=288 cells, doxorubicin=280 cells, palbociclib=276 cells, abemaciclib=266 cells from 3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant. Doxo, doxorubicin. Abe, abemaciclib. dpt, days post treatment.


( B ) BJ cells transduced with a NF - κB reporter were treated with vehicle , doxorubicin , paclitaxel ( 50 nM for 24 hours ) , palbociclib ( 1 μM for 8 times 24 hours ) or abemaciclib and luciferase activity measured 8 dpt . TNFα treatment was used as positive control ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant .

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

(B) BJ cells transduced with a NF-κB reporter were treated with vehicle, doxorubicin, paclitaxel (50 nM for 24 hours), palbociclib (1 μM for 8 times 24 hours) or abemaciclib and luciferase activity measured 8 dpt. TNFα treatment was used as positive control (n=3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.


( E ) 8 dpt , qRT - PCR of indicated NF - κB associated SASP genes was performed using indicated treated BJ cells 8 dpt ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant .

(E) 8 dpt, qRT-PCR of indicated NF-κB associated SASP genes was performed using indicated treated BJ cells 8 dpt (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.


( G ) Proteins in the serum - free conditioned media ( CM ) collected from doxorubicin - or abemaciclib - treated BJ fibroblasts ( 8 dpt ) were measured by cytokine array . Purple boxes indicated internal positive controls from each membrane ; Red boxes highlighted the most typical SASP factors .

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

(G) Proteins in the serum-free conditioned media (CM) collected from doxorubicin- or abemaciclib-treated BJ fibroblasts (8 dpt) were measured by cytokine array. Purple boxes indicated internal positive controls from each membrane; Red boxes highlighted the most typical SASP factors.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . RNA isolated from kidneys and mRNA encoding indicated NF - κB associated genes ( J ) quantified by qRT - PCR . n = 6 mice / group . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). RNA isolated from kidneys and mRNA encoding indicated NF-κB associated genes (J) quantified by qRT-PCR. n=6 mice/group. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . From a subset of the mice , RFP + cells were sorted from the renal cortex using FACS and mRNA encoding indicated NF - κB associated pro - inflammatory genes quantified by qRT - PCR . The values of fold change over vehicle were plotted in the heatmap . n = 4 mice / group ( K ) .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). From a subset of the mice, RFP+ cells were sorted from the renal cortex using FACS and mRNA encoding indicated NF-κB associated pro-inflammatory genes quantified by qRT-PCR. The values of fold change over vehicle were plotted in the heatmap. n=4 mice/group (K).


( B ) Human BJ fibroblasts , hTERT - RPE1 epithelial cells and lung mesenchymal stem cells were treated with vehicle ( DMSO for 8 times 24 hours ) or doxorubicin ( 250 nM for 24 hours ) or paclitaxel ( 50 nM for 24 hours ) or palbociclib ( 1 μM for 8 times 24 hours ) or abemaciclib ( 1 μM for 8 times 24 hours ) . At 8 dpt , RNA was isolated and mRNA for the indicated p53 - associated SASP genes quantified by qRT - PCR relative to tubulin ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B) Human BJ fibroblasts, hTERT-RPE1 epithelial cells and lung mesenchymal stem cells were treated with vehicle (DMSO for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) or paclitaxel (50 nM for 24 hours) or palbociclib (1 μM for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). At 8 dpt, RNA was isolated and mRNA for the indicated p53-associated SASP genes quantified by qRT-PCR relative to tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( D and E ) RNA was isolated from vehicle , abemaciclib ( 1 μM ) ( D ) or palbociclib ( 1 μM ) ( E ) treated scramble / shp53 BJ cells and quantified by qRT - PCR for IGFBP3 or LIF genes ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D and E) RNA was isolated from vehicle, abemaciclib (1 μM) (D) or palbociclib (1 μM) (E) treated scramble/shp53 BJ cells and quantified by qRT-PCR for IGFBP3 or LIF genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( F ) RNA isolated from kidneys treated with doxorubicin or abemaciclib and mRNA encoding indicated p53 - associated SASP genes quantified by qRT - PCR ( n = 6 mice / group ) . ( G ) RNA was isolated from kidneys of p16 - 3MR mice treated with abemaciclib ( 50 mg / kg , 7 consecutive days ) ± pifithrin - α ( 2 mg / kg , 7 consecutive days ) , and mRNA encoding p53 associated SASP genes was quantified by qRT - PCR and normalized on tubulin ( n = 5 mice / group ) . ( Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(F) RNA isolated from kidneys treated with doxorubicin or abemaciclib and mRNA encoding indicated p53-associated SASP genes quantified by qRT-PCR (n=6 mice/group). (G) RNA was isolated from kidneys of p16-3MR mice treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days), and mRNA encoding p53 associated SASP genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). ( Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( I ) RNA was isolated from vehicle or nutlin - 3a ( 1 μM for 8 times 24 hours ) treated BJ cells at 8 dpt and quantified by qRT - PCR for p53 target cell cycle genes , SASP genes ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(I) RNA was isolated from vehicle or nutlin-3a (1 μM for 8 times 24 hours) treated BJ cells at 8 dpt and quantified by qRT-PCR for p53 target cell cycle genes, SASP genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( J ) Mouse dermal fibroblasts ( MDF ) or MDF - p53 - / - fibroblasts were transfected with empty vector ( EV ) or p53 - overexpressing vector ( p53OE ) , then the cells were treated with vehicle ( water ) or doxorubicin ( 250 nM for 24 hours ) and qRT - PCR was performed for Igfbp3 and Lif genes ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . EV , empty

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

(J) Mouse dermal fibroblasts (MDF) or MDF-p53-/- fibroblasts were transfected with empty vector (EV) or p53-overexpressing vector (p53OE), then the cells were treated with vehicle (water) or doxorubicin (250 nM for 24 hours) and qRT-PCR was performed for Igfbp3 and Lif genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. EV, empty vector


( A ) MCF7 cells were incubated with serum - free CM collected from treated BJ fibroblasts ( 8 dpt ) containing EdU for 45 hours and EdU + cells quantified ( n = 9 samples from 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant . CM , conditioned media .

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

(A) MCF7 cells were incubated with serum-free CM collected from treated BJ fibroblasts (8 dpt) containing EdU for 45 hours and EdU+ cells quantified (n=9 samples from 3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. CM, conditioned media.


( B ) MCF7 cells migrated through the pores of trans - well after 24 hours incubation with CM ( 8 dpt ; scale bar , 1 mm ; n = 9 samples from 3 independent experiments ) were stained with 0 . 2 % crystal violet and quantified . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant . CM , conditioned media .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MCF7 cells migrated through the pores of trans-well after 24 hours incubation with CM (8 dpt; scale bar, 1 mm; n=9 samples from 3 independent experiments) were stained with 0.2% crystal violet and quantified. Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. CM, conditioned media.


( H and I ) 2 * 105 cells of vehicle - treated ( water for 8 times 24 hours ) mouse dermal fibroblasts ( MDF ) were injected into the left flank of the p16 - 3MR mice . Doxorubicin - induced ( 250 nM for 24 hours ) ( H ) or abemaciclib - induced ( 4 μM for 8 times 24 hours ) ( I ) MDFs were injected into the right flank of the same animal . 7 dpt , the above - mentioned mice were injected with coelenterazine and bioluminescence from the p16 - 3MR mouse was visualized / quantified by the IVIS spectrum in vivo imaging system and quantified . Lower panel , scheme of experimental design ( n = 4 mice / group ) . Data information : Data are means ± SD . Paired student t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

(H and I) 2*105 cells of vehicle-treated (water for 8 times 24 hours) mouse dermal fibroblasts (MDF) were injected into the left flank of the p16-3MR mice. Doxorubicin-induced (250 nM for 24 hours) (H) or abemaciclib-induced (4 μM for 8 times 24 hours) (I) MDFs were injected into the right flank of the same animal. 7 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the p16-3MR mouse was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified. Lower panel, scheme of experimental design (n=4 mice/group). Data information: Data are means ±SD. Paired student t-test *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


( J ) Equal amount ( 2 * 105 cells ) of doxorubicin - induced ( 250 nM for 24 hours , right flank ) and abemaciclib - induced ( 4 μM for 8 times 24 hours , left flank ) senescent MDF - 3MR cells were subcutaneously injected into the same wild - type mice . At 1 dpt , 7 dpt and 15 dpt , the above - mentioned mice were injected with coelenterazine and bioluminescence from the injected MDF - 3MR cells was visualized / quantified by the IVIS spectrum in vivo imaging system and quantified ( n = 4 mice / group ) . Upper panel , scheme of experimental design . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

(J) Equal amount (2*105 cells) of doxorubicin-induced (250 nM for 24 hours, right flank) and abemaciclib-induced (4 μM for 8 times 24 hours, left flank) senescent MDF-3MR cells were subcutaneously injected into the same wild-type mice. At 1 dpt, 7 dpt and 15 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the injected MDF-3MR cells was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified (n=4 mice/group). Upper panel, scheme of experimental design. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


C , Growth curves for E . coli 113 / 3 in defined media and in the presence or absence of B12 ( and o , r espectively ) . When challenged with 40 nM isolated R - domain ( ) , cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A ( ▫ ) a pproached that of the no colicin control , consistent with OmpF threading causing the disengagement of ColE9 from BtuB . Error bars represent the standard deviation across three biological replicates .

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

C, Growth curves for E. coli 113/3 in defined media and in the presence or absence of B12 ( and o, respectively). When challenged with 40 nM isolated R-domain ( ), cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A (▫) approached that of the no colicin control, consistent with OmpF threading causing the disengagement of ColE9 from BtuB. Error bars represent the standard deviation across three biological replicates.


ColE9 * denotes ColE9 H551A , an active site mutation that inactivates the toxin . A , Widefield fluorescence microscopy images of E . coli JM83 cells labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) and Hoechst DNA stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment with or without prior treatment with CCCP . Each panel shows the same cell in DIC ( grey ) , Hoechst DNA stain ( blue ) and ColE9 * K469CAF647 fluorescence ( red ) . Overlays of Hoechst and ColE9 * K469CAF647 fluorescence are also shown . ColE9 * K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin . Trypsin treatment in the absence of CCCP yields some cell - associated ColE9 * K469CAF647 fluorescence that likely represents internalised molecules ( see text ) . Scale bar , 1 µm .

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

ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.


C , Box and whisker plots showing mean pixel intensities for ColE9 * K469CAF647 fluorescence per cell measured for the indicated cells and condition used , whiskers represent minimum and maximum mean pixel intensity , box shows 1st and 3rd quartile with the median shown as a line . From left - to - right : E . coli JM83 cells ; E . coli btuB deletion strain showing loss of all ColE9 * K469CAF647 cell - associated fluorescence ; E . coli JM83 cells in the presence of CCCP ; E . coli JM83 cells following trypsin treatment showing significant ColE9 * K469CAF647 fluorescence remains associated with cells indicative of import ; E . coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9 * K469CAF647 fluorescence . n , number of cells used , typically from 2 - 4 biological replicates . * * * * , indicates a t value < 0 . 05 in a student T - test as a statistically significant result , ns indicates no significant difference as determined by student T - test .

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

C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.


B , Overnight plate assays comparing the cytotoxic activity of wild - type ColE9 with ColE9 K469C labelled with AF488 , AF568 or AF647 against a lawn of E . coli with either OmpF ( JW2203 ) or OmpC ( JW0912 ) in the outer membrane ( shown as + OmpF and + OmpC , respectively ) . Each plate was spotted with a serial dilution of the colicin ( 10 µM - 2 . 4 pM , 4 - fold dilution per spot ) . Wild - type ColE9 was more active against E . coli cells with OmpF in the outer membrane than OmpC . Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Overnight plate assays comparing the cytotoxic activity of wild-type ColE9 with ColE9 K469C labelled with AF488, AF568 or AF647 against a lawn of E. coli with either OmpF (JW2203) or OmpC (JW0912) in the outer membrane (shown as +OmpF and +OmpC, respectively). Each plate was spotted with a serial dilution of the colicin (10 µM - 2.4 pM, 4-fold dilution per spot). Wild-type ColE9 was more active against E. coli cells with OmpF in the outer membrane than OmpC. Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC.


C , Widefield fluorescence microscopy images of E . coli JW2203 ( + OmpF ) and JW0912 ( + OmpC ) cells labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) and Hoechst stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment . Each panel shows the same cell in DIC ( grey ) , Hoechst DNA stain ( blue ) and fluorescence of ColE9 * K469CAF647 ( red ) . Overlays of Hoechst and ColE9 * K469CAF647 fluorescence are also shown . Data show that following trypsin - treatment , significant ColE9 * K469CAF647 fluorescence remains associated with ompF - expressing E . coli whereas little or no fluorescence remains associated with ompC - expressing E . coli . Scale bar , 1 µm . D , Box and whisker plots of E . coli JW2203 and E . coli JW0912 cells labelled with ColE9 * K469CAF647 with and without trypsin treatment : whiskers represent minimum and maximum mean pixel intensity , box shows 1st and 3rd quartile with the median shown as a line . Microscopy data were collected as in c . The mean pixel intensity of ColE9 * K469CAF647 per cell was measured for each cell condition . n , number of cells , typically from 3 or 4 biological replicates . Data show that ColE9 * K469CAF647 translocates across the OM through OmpF and that this is impeded when E . coli has OmpC in the outer membrane .

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

C, Widefield fluorescence microscopy images of E. coli JW2203 (+OmpF) and JW0912 (+OmpC) cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst stain (20 µM) for 30 min at 37 °C with or without trypsin treatment. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and fluorescence of ColE9* K469CAF647 (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. Data show that following trypsin-treatment, significant ColE9* K469CAF647 fluorescence remains associated with ompF-expressing E. coli whereas little or no fluorescence remains associated with ompC-expressing E. coli. Scale bar, 1 µm. D, Box and whisker plots of E. coli JW2203 and E. coli JW0912 cells labelled with ColE9* K469CAF647 with and without trypsin treatment: whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. Microscopy data were collected as in c. The mean pixel intensity of ColE9* K469CAF647 per cell was measured for each cell condition. n, number of cells, typically from 3 or 4 biological replicates. Data show that ColE9* K469CAF647 translocates across the OM through OmpF and that this is impeded when E. coli has OmpC in the outer membrane.


A , B Co - immunoprecipitation ( IP ) of EBNA2 ( A ) and PLK1 ( B ) using EBNA2 - , PLK1 - specific antibodies , or isotype - matched control antibodies performed on total cell lysates ( L ) of lymphoblastoid long - term B cell cultures ( LCLs ) infected with recombinant EBV strain 2089 , which is based on EBV strain B95 -

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A, B Co-immunoprecipitation (IP) of EBNA2 (A) and PLK1 (B) using EBNA2-, PLK1-specific antibodies, or isotype-matched control antibodies performed on total cell lysates (L) of lymphoblastoid long-term B cell cultures (LCLs) infected with recombinant EBV strain 2089, which is based on EBV strain B95-8


GFP - tagged EBNA2 fragments to co - precipitate endogenous PLK1 . Total protein lysates and immunoprecipitates ( IP ) were analyzed by Western blotting ( WB ) .

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GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB).


( H ) Co - immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1 .

(H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.


( D ) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co - immunoprecipitations followed by Western blotting .

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(D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting.


( F ) GST EBNA2 fragment 246 - 487 wt and mutant S457A / T465V were treated with recombinant active PLK1 ( + ) in the presence of [ γ - 32P ] ATP in vitro or left untreated ( - ) . CRS , an artificial PLK1 test substrate was used as a positive control .

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(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate was used as a positive control.


( H ) HA - tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2 / CBF1 responsive promoter reporter luciferase plasmid . Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity .

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

(H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity.


( A ) Flag - tagged active ( PLK1 ) and kinase inactive PLK1 ( K82M ) were co - expressed with HA - EBNA2 wt , docking site mutant ( S379A ) and phosphorylation mutant ( S457A / T465V ) and tested for activation of an EBNA2 / CBF1 responsive promoter reporter in DG75 cells . Activation of the reporter firefly luciferase gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to EBNA2 wt activity .

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

(A) Flag-tagged active (PLK1) and kinase inactive PLK1 (K82M) were co-expressed with HA-EBNA2 wt, docking site mutant (S379A) and phosphorylation mutant (S457A/T465V) and tested for activation of an EBNA2/CBF1 responsive promoter reporter in DG75 cells. Activation of the reporter firefly luciferase gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to EBNA2 wt activity.


( B ) 30 µg of cellular extracts that were produced for luciferase assays were tested for protein expression using Flag - , HA - and GAPDH - specific antibodies .

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(B) 30 µg of cellular extracts that were produced for luciferase assays were tested for protein expression using Flag-, HA- and GAPDH-specific antibodies.


( C ) Expression of EBNA2 , LMP1 , PLK1 , MYC , and GAPDH was analyzed by Western blotting and immunostaining of whole cell extracts of cell lines established from 3 individual donors .

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(C) Expression of EBNA2, LMP1, PLK1, MYC, and GAPDH was analyzed by Western blotting and immunostaining of whole cell extracts of cell lines established from 3 individual donors.


( B ) Survival curve of humanized mice infected with 105 GRU EBV wt ( n = 13 ) , EBV S379A ( n = 11 ) or EBV S457A / T465V EBV ( n = 9 ) .

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(B) Survival curve of humanized mice infected with 105 GRU EBV wt (n = 13), EBV S379A (n = 11) or EBV S457A/T465V EBV (n = 9).


( E ) Development of viral loads in blood of infected mice over a period of five weeks . Error bars indicate mean ± SEM . Number of biological replicates per group : EBNA2 WT EBV week 2 - 5 : n = 8 ; EBNA2 S379A EBV week 2 - 5 : n = 7 ; EBNA2 S457A T465V EBV week 2 : n = 9 , week 3 : n = 8 , week 4 and week 5 : n =

(E) Development of viral loads in blood of infected mice over a period of five weeks. Error bars indicate mean ± SEM. Number of biological replicates per group: EBNA2 WT EBV week 2 - 5: n = 8; EBNA2 S379A EBV week 2 - 5: n = 7; EBNA2 S457A T465V EBV week 2: n = 9, week 3: n = 8, week 4 and week 5: n = 7


( F ) Flow - cytometric analyses of CD8 + / CD4 + T cell ratios in the blood of infected mice over a period of five weeks . : EBNA2 WT EBV week 0 , 2 , 4 , 5 : n = 13 , week 3 : n = 8 ; EBNA2 S379A week 0 , 2 , 4 , 5 : n = 11 , week 3 : n = 7 ; EBNA2 S457A T465V week 0 , 2 : n = 12 , week 3 : n = 7 , week 4 : n = 10 , week 5 : n = 9 .

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

(F) Flow-cytometric analyses of CD8+/CD4+ T cell ratios in the blood of infected mice over a period of five weeks. : EBNA2 WT EBV week 0, 2, 4, 5: n = 13, week 3: n = 8 ; EBNA2 S379A week 0, 2, 4, 5: n = 11, week 3: n = 7 ; EBNA2 S457A T465V week 0, 2: n = 12, week 3: n = 7, week 4: n = 10, week 5: n = 9.


METTL14 is arginine methylated at its C - terminal IDR in cells . HEK293 cells expressing Flag - tagged FL or C - terminal IDR - truncated ( 1 - 400 ) METTL14 were lysed and immunoprecipitated with an anti - Flag antibody . Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA . the black triangles indicate arginine methylated - METTL14 ; open triangles indicate the immunoprecipitated METTL14 proteins .

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

METTL14 is arginine methylated at its C-terminal IDR in cells. HEK293 cells expressing Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14 were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins.


The C - terminal IDR of METTL14 is essential for its interaction with PRMT1 . HEK293 cells were transfected with GFP - tagged PRMT1 and Flag - tagged FL or C - terminal IDR - truncated ( 1 - 400 ) METTL14 . IP was performed using an anti - GFP antibody , and Western blot analysis was performed using anti - GFP and anti - Flag antibodies .

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

The C-terminal IDR of METTL14 is essential for its interaction with PRMT1. HEK293 cells were transfected with GFP-tagged PRMT1 and Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14. IP was performed using an anti-GFP antibody, and Western blot analysis was performed using anti-GFP and anti-Flag antibodies.


Knockdown of PRMT1 expression reduces METTL14 arginine methylation . HEK293 cells were transfected with control siRNA ( siCtrl ) and the siRNA targeting PRMT1 ( siPRMT1 ) . METTL14 was immunoprecipitated from these cells , and its methylation level was detected by Western blot analysis using an anti - ASYM26 antibody . * indicates the location of the IgG heavy chain .

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

Knockdown of PRMT1 expression reduces METTL14 arginine methylation. HEK293 cells were transfected with control siRNA (siCtrl) and the siRNA targeting PRMT1 (siPRMT1). METTL14 was immunoprecipitated from these cells, and its methylation level was detected by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain.