A , D Stably transfected MDA - MB - 231 cells bearing a control ( A ) , DAPK1 ( B ) , UNC5B ( C ) , or NTN1 shRNA ( D ) were injected into the mammary fat pad of immuno - compromised mice . When tumors reached 100 - 120 mm3 , mice were injected subcutaneously with DAC ( 0 . 4 mg / kg ) or PBS and / or intraperitoneally with net1 - mAb ( 10 mg / kg ) . Tumor volumes were measured twice a week , n = 8 mice per group . The statistical significance of the differences obtained between DAC - group and DAC + net1 - mAb - group for shControl , DAC - group and DAC + net1 - mAb - group for shDAPK1 and shUNC5B , and PBS - group and DAC - group for shNTN1 , respectively , were determined by two - way ANOVA and a post - hoc Tukey - test . * * * * P < 0 . 0001 , , ns = not significant . Error bars = s . e . m .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-ORGANISM O O B-TISSUE O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, D Stably transfected MDA-MB-231 cells bearing a control (A), DAPK1 (B), UNC5B (C), or NTN1 shRNA (D) were injected into the mammary fat pad of immuno-compromised mice. When tumors reached 100-120 mm3, mice were injected subcutaneously with DAC (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). Tumor volumes were measured twice a week, n = 8 mice per group. The statistical significance of the differences obtained between DAC-group and DAC + net1-mAb-group for shControl, DAC-group and DAC + net1-mAb-group for shDAPK1 and shUNC5B, and PBS-group and DAC-group for shNTN1, respectively, were determined by two-way ANOVA and a post-hoc Tukey-test. **** P<0.0001, , ns = not significant. Error bars = s.e.m.


A . Infection by a L . pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification . Raw264 . 7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h . Saponin soluble proteins resolved by SDS - PAGE were probed with a UBE2N - specific antibody ( top panel ) . The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins . Note that MavC but not MvcA is detectable in cells infected with wild type bacteria ( middle panels labeled as " translocation " ) . Tubulin was probed as a loading control . Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction ( lower panels labeled as " expression " ) . The bacterial metabolism enzyme isocitrate dehydrogenase ( ICDH ) was probed as a loading control . Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium ( the middle panel of the lower portion ) .

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

A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion).


B . Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells . HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA . 16 h after transfection , modification of endogenous UBE2N was detected by immunoblotting ( upper level ) . The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis ( Ypt ) , which cannot modify UBE2N , was included as a control . Note that coexpression of MvcA led to accumulation of unmodified UBE2N ( 4th lane ) in samples that expressed MavC . The expression of MavC MvcA and Cif was detected using a Flag - specific antibody ( lower panel ) . Tubulin was probed as a loading control ( lower panel ) .

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

B. Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC MvcA and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel).


C . MvcA interferes with MavC - induced UBE2N ubiquitination . Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS - PAGE were detected by Coomassie Brilliant blue ( CBB ) staining . Note that MvcA ( inclusion of MvcA with MavC , 7th lane . Compare to MavC alone in 3rd lane ) but not the MvcAC83A mutant ( 8th lane ) abolished MavC - induced UBE2N modification .

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

C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification.


D . Dose - dependent modification of UBE2N by MavC and MvcA . A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min . Samples resolved by SDS - PAGE were detected by CBB staining .

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

D. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min. Samples resolved by SDS-PAGE were detected by CBB staining.


A . The hydrolytic cleavage of UBE2N - Ub by MvcA into UBE2N and ubiquitin ( Q40E variant ) . Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS - PAGE were detected by CBB staining ( left ) . Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N - Ub ( right ) . The ubiquitin product is the Glu40 variant of ubiquitin .

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

A. The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin.


C . Dose - dependent cleavage of UBE2N - Ub by MvcA . A series of reactions containing UBE2N - Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS - PAGE and CBB staining . Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N - Ub and MvcA is 2048 : 1 .

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

C. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS-PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2048:1.


D . Time - dependent cleavage of UBE2N - Ub by MvcA . UBE2N - Ub was incubated with MvcA at the molar ratio of 128 . Samples taken at the indicated time points were resolved by SDS - PAGE and detected by CBB staining .

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

D. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS-PAGE and detected by CBB staining.


E . UBE2N produced from UBE2N - Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains . UBE2N - Ub was preincubated with MvcA or its inactive mutant for 30 min . Then a cocktail containing E1 , UBE2V2 , 3xHA - ubiquitin , the E3 enzyme TRAF6 and ATP was added into reactions . Ubiquitination was allowed to proceed for 30 min . Proteins in samples resolved by SDS - PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody . Note the robust formation of polyubiquitin chains in the reaction receiving MvcA ( middle lane ) .

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

E. UBE2N produced from UBE2N-Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was preincubated with MvcA or its inactive mutant for 30 min. Then a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6 and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane).


H . Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis . For deubiquitination reactions , the indicated amounts of MvcA or its mutants were incubated with 2 . 0 μg UBE2N for 5 min in 25 μl reactions . For ubiquitin deamidase activity , the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions . The ratio of cleavage was analyzed by SDS - PAGE and then densitometry . Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments . Bars and error bars : means and standard error of the mean ( SEM ) .

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

H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).


I - J . The roles of ubiquitin contact residues in the activity of MvcA . I . The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N - Ub for 5 min in 25 μl reactions . The ratio of cleavage was analyzed by SDS - PAGE and then densitometry . Each bar represents the ratio of cleavage obtained from three independent experiments . Bars and error bars : means and standard error of the mean ( SEM ) . J . The activity of the MvcA mutant harboring three mutations . 0 . 05 μg , 0 . 25 μg and 1 . 25 μg of MvcA or the mutant was incubated with 2 μg UBE2N - Ub in 25 μl reactions . After 30 min incubation , the products resolved by SDS - PAGE were detected by CBB staining . Note that the mutant did not detectable cleave UBE2N - Ub even in the reaction received the highest amount of protein .

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

I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.


A . Overexpression of MavC but not the inactive mutant MavCC74A in the ∆ mavC ∆ mvcA mutant background causes IκBα accumulation in infected cells . U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα . Tubulin was probed as a loading control .

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

A. Overexpression of MavC but not the inactive mutant MavCC74A in the ∆mavC∆mvcA mutant background causes IκBα accumulation in infected cells. U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα. Tubulin was probed as a loading control.


B . Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation . HEK293 cells transfected to express the FcγII receptor and 4xFlag - MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L . pneumophila that expresses GFP for 12 h . The vacuole was scored and categorized by the number of bacteria it contained . Data ( mean + / - SE ) shown were from three independent experiments , each with at least 300 vacuoles counted . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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

B. Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation. HEK293 cells transfected to express the FcγII receptor and 4xFlag-MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L. pneumophila that expresses GFP for 12 h. The vacuole was scored and categorized by the number of bacteria it contained. Data (mean +/- SE) shown were from three independent experiments, each with at least 300 vacuoles counted. *, p<0.05; **, p<0.01; n.s., not significant.


C . Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆ mavC ∆ mvcA mutant of L . pneumophila . Raw264 . 7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points . Each strain was examined in triplicate and the values shown were mean + / - SE . Similar results were obtained in two independent experiments . * , p < 0 . 1 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( t - test by comparing p ( MavC ) with p ( MavCC74A ) ) .

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

C. Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆mavC∆mvcA mutant of L. pneumophila. Raw264.7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Each strain was examined in triplicate and the values shown were mean +/- SE. Similar results were obtained in two independent experiments. *, p<0.1; **, p<0.01; ***, p<0.001(t-test by comparing p(MavC) with p(MavCC74A)).


D . MvcA is required for reversal of UBE2N modification during L . pneumophila infection . U937 cells infected with wild type or the ∆ mvcA mutant were withdrawn at the indicated time points . Samples resolved by SDS - PAGE were probed for UBE2N , translocated MavC and MvcA . Tubulin was probed as a loading control . Note that in samples infected with the ∆ mvcA strain , the level of UBE2N - Ub decreased at slower rates .

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

D. MvcA is required for reversal of UBE2N modification during L. pneumophila infection. U937 cells infected with wild type or the ∆mvcA mutant were withdrawn at the indicated time points. Samples resolved by SDS-PAGE were probed for UBE2N, translocated MavC and MvcA. Tubulin was probed as a loading control. Note that in samples infected with the ∆mvcA strain, the level of UBE2N-Ub decreased at slower rates.


E . MvcA becomes detectable in intracellular bacteria 5 h after uptake . U937 macrophages infected with wild type L . pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA , respectively . The bacterial isocitrate dehydrogenase ( ICDH ) was probed sequentially as a loading control .

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

E. MvcA becomes detectable in intracellular bacteria 5 h after uptake. U937 macrophages infected with wild type L. pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA, respectively. The bacterial isocitrate dehydrogenase (ICDH) was probed sequentially as a loading control.


F . MvcA restores the nuclear translocation of p65 inhibited by MavC during L . pneumophila infection . Raw264 . 7 cells were infected with the indicated L . pneumophila strains for 2 h at an MOI of 1 . Fixed cells were immunostained with antibodies specific for p65 and the bacterium , respectively . Host nuclei were labeled with Hoechst . Representative images of cells in each sample category were shown ( upper panels ) ; nuclear localization of p65 was determined by scoring at least 300 infected cells ( lower panel ) . Results shown were from three independent experiments done in triplicate and the values shown were mean + / - SE ; t - test .

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

F. MvcA restores the nuclear translocation of p65 inhibited by MavC during L. pneumophila infection. Raw264.7 cells were infected with the indicated L. pneumophila strains for 2 h at an MOI of 1. Fixed cells were immunostained with antibodies specific for p65 and the bacterium, respectively. Host nuclei were labeled with Hoechst. Representative images of cells in each sample category were shown (upper panels); nuclear localization of p65 was determined by scoring at least 300 infected cells (lower panel). Results shown were from three independent experiments done in triplicate and the values shown were mean +/- SE; t-test.


G . Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L . pneumophila infection . U937 cells were infected with the indicated bacterial strains at an MOI of 5 . Lysates of cells infected for the indicated time were probed for IκBα . Tubulin was probed as a loading control . IκBα levels for cells infected with the strains ∆ mvA ( pMvcA ) and ∆ mvA ( pMvcAC83A ) at the indicated time points were evaluated by measuring the band intensity from three independent experiments ( lower panel ) , the values shown were mean + / - SE ; t - test .

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G. Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L. pneumophila infection. U937 cells were infected with the indicated bacterial strains at an MOI of 5. Lysates of cells infected for the indicated time were probed for IκBα. Tubulin was probed as a loading control. IκBα levels for cells infected with the strains ∆mvA(pMvcA) and ∆mvA(pMvcAC83A) at the indicated time points were evaluated by measuring the band intensity from three independent experiments (lower panel), the values shown were mean +/- SE; t-test.


Live imaging of histone - GFP ( green ) and ParB - mCherry ( magenta ) expressed in larval salivary gland nuclei using 1151 - Gal4 . H3 . 3 - GFP levels are increased at the E ( spl ) - C in the presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . ParB - mCherry binds to its cognate int DNA sequence inserted within the E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C and yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( white ) = 5 µm . Quantifications of relative fluorescence intensity of histone - GFP and ParB - mCherry across the E ( spl ) - C in Notch - OFF ( upper ) and Notch - ON ( lower ) conditions . Mean + / - SEM ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 and nglands = 5 , 5 , 5 , 5 , 5 , 4 where each gland represents a biological replicate ( from top to bottom ) .

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Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. H3.3-GFP levels are increased at the E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) and ParB - mCherry ( magenta ) expressed in larval salivary gland nuclei using 1151 - Gal4 . E ( spl ) - C in the presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ but there is little change in H3 - GFP between Notch - OFF and Notch - ON nuclei ParB - mCherry binds to its cognate int DNA sequence inserted within the E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C and yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( white ) = 5 µm . Quantifications of relative fluorescence intensity of histone - GFP and ParB - mCherry across the E ( spl ) - C in Notch - OFF ( upper ) and Notch - ON ( lower ) conditions . Mean + / - SEM ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 and nglands = 5 , 5 , 5 , 5 , 5 , 4 where each gland represents a biological replicate ( from top to bottom ) .

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Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ but there is little change in H3-GFP between Notch-OFF and Notch-ON nuclei ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) and ParB - mCherry ( magenta ) expressed in larval salivary gland nuclei using 1151 - Gal4 . E ( spl ) - C in the presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . with H3 . 3core - GFP ParB - mCherry binds to its cognate int DNA sequence inserted within the E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C and yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( white ) = 5 µm . Quantifications of relative fluorescence intensity of histone - GFP and ParB - mCherry across the E ( spl ) - C in Notch - OFF ( upper ) and Notch - ON ( lower ) conditions . Mean + / - SEM ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 and nglands = 5 , 5 , 5 , 5 , 5 , 4 where each gland represents a biological replicate ( from top to bottom ) .

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Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). with H3.3core-GFP ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Effects from depleting chromatin remodelers and histone chaperones , as indicated ( wide range shown in A , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary glands . Different OSA RNAi stocks used in C are denoted by ( 1 ) and ( 2 ) . In all conditions except Moira RNAi ( A ) nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . Percentage of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when the indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 glands were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed ; p < 0 . 0001 , two - tailed Fisher ' s exact test calculated using the raw ( non - percentage ) scoring data .

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Effects from depleting chromatin remodelers and histone chaperones, as indicated (wide range shown in A, on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. Different OSA RNAi stocks used in C are denoted by (1) and (2). In all conditions except Moira RNAi (A) nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C ) , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary glands . w RNAi is a control and BrmK804R is expression of dominant - negative Brm . Different OSA RNAi stocks used in C are denoted by ( 1 ) and ( 2 ) . , Snr1 RNAi and BrmK804R expression ( C ) , nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . Percentage of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when the indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 glands were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed ; p < 0 . 0001 , two - tailed Fisher ' s exact test calculated using the raw ( non - percentage ) scoring data .

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Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C), on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. w RNAi is a control and BrmK804R is expression of dominant-negative Brm. Different OSA RNAi stocks used in C are denoted by (1) and (2). , Snr1 RNAi and BrmK804R expression (C), nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


Chromatin accessibility in Notch - OFF ( B ) and Notch - ON ( NΔECD expression , C ) salivary gland nuclei measured by ATAC - qPCR ; fold enrichment at the indicated regions compared to a " closed ctrl " region . Expression of dominant - negative Brm , BrmK804R , led to reduced accessibility of E ( spl ) mβ - HLH and E ( spl ) m3 - HLH enhancer regions in Notch - OFF conditions , and to a more widespread reduction in accessibility in Notch - ON conditions . " Eip78C enh " corresponds to the ecdysone receptor - binding region of the Eip78C enhancer , which is highly accessible but not Notch - responsive ; " Rab11 tr " and " Mst87F tr " represent highly and lowly - expressed control genes respectively . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with two - tailed Welch ' s t - test comparing LacZ and BrmK804R samples .

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Chromatin accessibility in Notch-OFF (B) and Notch-ON (NΔECD expression, C) salivary gland nuclei measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Expression of dominant-negative Brm, BrmK804R, led to reduced accessibility of E(spl)mβ-HLH and E(spl)m3-HLH enhancer regions in Notch-OFF conditions, and to a more widespread reduction in accessibility in Notch-ON conditions. "Eip78C enh" corresponds to the ecdysone receptor-binding region of the Eip78C enhancer, which is highly accessible but not Notch-responsive; "Rab11 tr" and "Mst87F tr" represent highly and lowly-expressed control genes respectively. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test comparing LacZ and BrmK804R samples.


Chromatin accessibility in salivary gland nuclei depleted for Su ( H ) by RNAi , measured by ATAC - qPCR ; fold enrichment at the indicated regions compared to a " closed ctrl " region . Accessibility is increased across most of the E ( spl ) - C compared to controls expressing LacZ . Control primer regions are as in B and C . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with two - tailed Welch ' s t - test compared to LacZ controls .

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Chromatin accessibility in salivary gland nuclei depleted for Su(H) by RNAi, measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Accessibility is increased across most of the E(spl)-C compared to controls expressing LacZ. Control primer regions are as in B and C. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test compared to LacZ controls.


Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively , measured by reverse transcription - qPCR in Kc167 cells ; percentage cDNA compared to GFP RNAi . The knockdowns are highly effective , with only 1 - 2 % of brm and Snr1 cDNA remaining detectable . Mean + / - SEM ; n = 3 .

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Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively, measured by reverse transcription-qPCR in Kc167 cells; percentage cDNA compared to GFP RNAi. The knockdowns are highly effective, with only 1-2% of brm and Snr1 cDNA remaining detectable. Mean +/- SEM; n = 3.


Knockdown of components of the BRM complex reduces Su ( H ) recruitment both in Notch - OFF ( B ) and Notch - ON ( C ) conditions . Fold enrichment of Su ( H ) occupancy at the indicated positions detected by ChIP , relative to input , in Kc167 cells treated with brm , Snr1 or GFP RNAi as a control . Notch - ON conditions ( C ) were induced by 30 minutes of EGTA treatment . Mean + / - SEM , n = 3 ( B ) ; Mean , n = 2 ( C ) ; * p < 0 . 05 with one - tailed student ' s t - test compared to GFP RNAi control .

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Knockdown of components of the BRM complex reduces Su(H) recruitment both in Notch-OFF (B) and Notch-ON (C) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with brm, Snr1 or GFP RNAi as a control. Notch-ON conditions (C) were induced by 30 minutes of EGTA treatment. Mean +/- SEM, n = 3 (B); Mean, n = 2 (C); * p<0.05 with one-tailed student's t-test compared to GFP RNAi control.


Effect of brm RNAi on E ( spl ) mβ - HLH ( mβ ) and E ( spl ) m3 - HLH ( m3 ) induction by Notch activation ( EGTA treatment ) measured by reverse transcription - qPCR ; shown as fold difference to lacZ RNAi control . Mean + / - SEM ; n = 3 .

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Effect of brm RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) induction by Notch activation (EGTA treatment) measured by reverse transcription-qPCR; shown as fold difference to lacZ RNAi control. Mean +/- SEM; n = 3.


Effect of Brm dominant - negative on expression of E ( spl ) mβ - HLH ( mβ ) and E ( spl ) m3 - HLH ( m3 ) measured by reverse transcription - qPCR . Expression was analyzed in stable cell lines containing pMT - inducible BrmWT or BrmK804R in the absence ( left , uninduced ) or presence of copper sulfate ( right , Cu2 + induced ) . The response of E ( spl ) mβ - HLH and E ( spl ) m3 - HLH to Notch activation ( " N - On " = EGTA treatment vs . " N - Off " = PBS control ) was reduced in the BrmK804R - expressing cells compared to BrmWT - expressing cells , only when induced with copper ( right graph ) . Mean , n = 2 ( left ) ; Mean + / - SEM , n = 3 ( right ) ; * p < 0 . 05 with one - tailed student ' s t - test comparing BrmWT and BrmK804R .

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

Effect of Brm dominant-negative on expression of E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) measured by reverse transcription-qPCR. Expression was analyzed in stable cell lines containing pMT-inducible BrmWT or BrmK804R in the absence (left, uninduced) or presence of copper sulfate (right, Cu2+ induced). The response of E(spl)mβ-HLH and E(spl)m3-HLH to Notch activation ("N-On" = EGTA treatment vs. "N-Off" = PBS control) was reduced in the BrmK804R-expressing cells compared to BrmWT-expressing cells, only when induced with copper (right graph). Mean, n = 2 (left); Mean +/- SEM, n = 3 (right); * p<0.05 with one-tailed student's t-test comparing BrmWT and BrmK804R.


Knockdown of actin - related subunit , BAP55 , reduces Su ( H ) recruitment in both Notch - OFF ( PBS treatment ) and Notch - ON ( EGTA treatment ) conditions . Fold enrichment of Su ( H ) occupancy at the indicated positions detected by ChIP , relative to input , in Kc167 cells treated with Bap55 or lacZ RNAi as a control . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with one - tailed student ' s t - test compared to lacZ RNAi control .

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

Knockdown of actin-related subunit, BAP55, reduces Su(H) recruitment in both Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with Bap55 or lacZ RNAi as a control. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Effect of Bap55 RNAi on E ( spl ) mβ - HLH ( mβ ) and E ( spl ) m3 - HLH ( m3 ) expression levels measured by reverse transcription - qPCR in Notch - OFF ( PBS treatment ) and Notch - ON ( EGTA treatment ) conditions . Expression level of RpII215 is shown as a control gene . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with one - tailed student ' s t - test compared to lacZ RNAi control .

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

Effect of Bap55 RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) expression levels measured by reverse transcription-qPCR in Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Expression level of RpII215 is shown as a control gene. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Nucleosome turnover measured by CATCH - IT - qPCR ; fold enrichment over input samples compared to Sec15 tr control region . Su ( H ) - bound enhancers show increased nucleosome turnover in response to Notch signaling . Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT - NICD with copper excluded in the control . Positions of E ( spl ) - C primers are shown in Fig 3A ; the remaining primers are control non - Notch - responsive regions . Mean , n = 2 .

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

Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2.


brm RNAi reduces nucleosome turnover at Notch - responsive regions . CATCH - IT - qPCR results as in A after brm or lacZ RNAi as a control . Mean + / - SEM ; n = 5 ; * p < 0 . 05 with one - tailed student ' s t - test comparing brm and lacZ RNAi .

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

brm RNAi reduces nucleosome turnover at Notch-responsive regions. CATCH-IT-qPCR results as in A after brm or lacZ RNAi as a control. Mean +/- SEM; n = 5; * p<0.05 with one-tailed student's t-test comparing brm and lacZ RNAi.


Brm is required for Notch - responsive nucleosome turnover . CATCH - IT - qPCR results after brm or lacZ RNAi as in B and pMT - NICD expression as in A . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with two - tailed student ' s t - test compared to control ( lacZ RNAi Notch - ON bars are compared to lacZ RNAi Notch - OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars ) .

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

Brm is required for Notch-responsive nucleosome turnover. CATCH-IT-qPCR results after brm or lacZ RNAi as in B and pMT-NICD expression as in A. Mean +/- SEM; n=3; * p<0.05 with two-tailed student's t-test compared to control (lacZ RNAi Notch-ON bars are compared to lacZ RNAi Notch-OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars).


brm RNAi reduces incorporation of histone H3 . 3 . V5 ChIP - qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3 - V5 or H3 . 3 - V5 expression induced from the pMT promoter by 3 hours of copper treatment , shown as fold enrichment over input samples . Mean + / - SEM ; n = 3 . * p < 0 . 05 with two - tailed Welch ' s t - test compared to lacZ RNAi control .

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

brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.


( B ) Epg5 protein and the wild - type copy of Epg5 are absent in Epg5 − / − mice . There is an intervening lane between Epg5 + / − and Epg5 − / − lanes in the Western blot .

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(B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot.


( C ) Body weight curves of Epg5 + / − and Epg5 − / − males . Means ± SEM of 12 mice are shown .

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(C) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown.


( D ) Time spent on a rotarod . The horizontal lines indicate mean times .

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(D) Time spent on a rotarod. The horizontal lines indicate mean times.


( E ) Epg5 − / − males show gradually worsening limb - clasping reflexes .

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(E) Epg5−/− males show gradually worsening limb-clasping reflexes.


( F ) The hindquarters of Epg5 − / − mice are completely paralyzed at 10 mo of age .

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(F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age.


( G ) Spinal deformation in an Epg5 − / − male at 10 mo of age .

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(G) Spinal deformation in an Epg5−/− male at 10 mo of age.


( H ) Epg5 − / − females gradually develop the limb - clasping phenotype .

(H) Epg5−/− females gradually develop the limb-clasping phenotype.


( A ) Nissl staining of the fifth layer of the cortex in 10 - mo - old Epg5 + / − and Epg5 − / − mice . ( B ) The number of pyramidal cells in the fifth layer per millimeters squared .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O

(A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared.


( C and D ) H & amp ; amp ; E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5 − / − mice . Means ± SEM of three mice are shown in B and D .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O

(C and D) H&amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D.


( E ) The normally developed cerebellum in Epg5 − / − mice . ( F ) The thickness of the molecular layer in the cerebellum in mutant and control mice .

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(E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice.


( G and H ) Purkinje cells , stained by calbindin , are not reduced in number in Epg5 − / − mice . Means ± SEM of five mice are shown in F and H .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O

(G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H.


( I ) Arrows indicate Pyknotic neurons in the anterior horn of Epg5 − / − mice .

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(I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/−mice.


( J ) The number of motor neurons in the spinal cord ( SC ) in Epg5 + / − and Epg5 − / − mice .

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(J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/−mice.


( K ) The numbers of Nissl - stained interneurons in the spinal cord are similar in control and Epg5 − / − mice . Means ± SEM of three mice are shown in J and K .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O

(K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K.


( L ) Eosinophilic spheroids ( arrows ) accumulate in the thoracic spinal cord of Epg5 − / − mice .

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(L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice.


( M ) Eosinophilic spheroids are swollen degenerating axons ( arrow ) .

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(M) Eosinophilic spheroids are swollen degenerating axons (arrow).


( N ) EM pictures showing degenerated axons ( arrows ) in the spinal cord of Epg5 − / − mice .

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(N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice.


( O and P ) GFAP staining in Epg5 + / − and Epg5 − / − mice . DCST , dorsal corticospinal tract .

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(O and P) GFAP staining in Epg5+/− and Epg5−/−mice. DCST, dorsal corticospinal tract.


( A ) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5 + / − and Epg5 − / − mice . SC , spinal cord .

O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O B-TISSUE I-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O B-TISSUE I-TISSUE O

(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/− mice. SC, spinal cord.


( B ) Polyubiquitinated proteins in detergent - soluble ( S ) and - insoluble ( I ) fractions from brain and spinal cord homogenates of Epg5 + / − and Epg5 − / − mice .

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(B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5+/− and Epg5−/− mice.


( C ) p62 aggregates and ubiquitin - positive aggregates are absent from Epg5 + / − mice but dramatically accumulate and are colocalized ( arrows ) in Epg5 − / − mice .

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(C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5−/− mice.


( D - F ) p62 aggregates are detected in neurons ( arrows ; D ) and oligodendrocytes ( arrows ; E ) but absent from astrocytes ( F ) in Epg5 − / − mice .

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(D-F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5−/− mice.


( G ) Cytoplasmic TDP - 43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5 − / − mice . Arrows show cytoplasmic TDP - 43 aggregates . DSCT , dorsal corticospinal tract . ( H ) Percentages of cytoplasmic TDP - 43 aggregate - positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5 + / − and Epg5 − / − mice . Means ± SEM of three mice are shown . LC , lateral column . Bars , 10 µm .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O

(G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.


( A and B ) Electromyography of the gastrocnemius muscle from a 3 - mo - old Epg5 − / − mouse showing fibrillations ( A ) and positive sharp waves ( B ) . These defects were absent in control mice .

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(A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice.


( C ) At 10 mo , Epg5 − / − mice showed high - amplitude and long - duration action potentials when conducting MUAP tests . The blue lines show the start and end of an action potential . Dur , duration ; Amp , amplitude .

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(C) At 10 mo, Epg5−/−mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude.


( D - F ) H & amp ; amp ; E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5 − / − mice . The arrow in E indicates centrally nucleated fibers .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-TISSUE O

(D-F) H&amp;E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5−/−mice. The arrow in E indicates centrally nucleated fibers.


( G ) The transcription levels of the atrophy - related genes were up - regulated in gastrocnemius muscles of Epg5 − / − mice .

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(G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5−/−mice.


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(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/−mice. SC, spinal cord.


( J ) Levels of LC3 - II and p62 in muscle extracts from Epg5 + / − and Epg5 − / − mice .

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(J) Levels of LC3-II and p62 in muscle extracts from Epg5+/− and Epg5−/− mice.


( K ) Percentage of p62 aggregate - positive myofibers in Epg5 + / − and Epg5 − / − mice . Means ± SEM of three mice are shown .

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(K) Percentage of p62 aggregate-positive myofibers in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown.


( L ) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5 − / − mice but not controls . Bars : ( D - F and L ) 10 µm ; ( H and I ) 1 µm .

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(L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5−/− mice but not controls. Bars: (D-F and L) 10 µm; (H and I) 1 µm.


( A ) Number of LC3 puncta in Epg5 + / − ( control ) and Epg5 − / − MEFs .

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(A) Number of LC3 puncta in Epg5+/− (control) and Epg5−/− MEFs.


( B ) Western blot showing levels of LC3 - I and LC3 - II in control and Epg5 − / − MEFs upon indicated treatment ( Starv . , starvation ; Rapa . , rapamycin ; Bafilo . , bafilomycin A1 ) .

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

(B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1).


( C ) Proteinase K ( ProK ) protection assay in different MEFs . PNS , postnuclear supernatant ; P , pellet fractions .

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

(C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions.


( D ) Colocalization ratio of LC3 and LAMP - 1 in control and Epg5 − / − MEFs . 50 cells were examined per time point in A and D .

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

(D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5−/− MEFs. 50 cells were examined per time point in A and D.


( E ) Colocalization of LC3 puncta and lysosomes in control and Epg5 − / − MEFs 4 h after starvation .

O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O B-CELL_LINE O O O O O

(E) Colocalization of LC3 puncta and lysosomes in control and Epg5−/− MEFs 4 h after starvation.


( F ) Under nutrient repletion conditions , almost no autophagic elements are detected in Epg5 + / − MEFs . RER , rough ER ; M , mitochondrion ; N , nucleus . The arrowhead indicates a vacuole with a late residual body - like appearance ( aAV - III ) . ( G ) Under nutrient repletion conditions , Epg5 − / − MEFs accumulate a large number of autophagic vacuoles . Red arrow , a likely autophagosome ; red arrowheads , complex vacuoles of the aAV - I type ; white arrowheads , aAV - II

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

(F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles


( H and I ) High magnification of autophagic elements in unstarved Epg5 − / − MEFs . ( H ) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures ( white arrowheads ) and a lipid - like region ( L ) . ( I ) The red arrow indicates an autophagosome containing cytoplasm and ribosomes . The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures . The white arrow shows an aAV - III vacuole .

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

(H and I) High magnification of autophagic elements in unstarved Epg5−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole.


( J ) Compared with control cells , Rab5 - labeled early endosomes are significantly enlarged in Epg5 siRNA - treated cells .

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(J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells.


( K and L ) EGFR degradation in control and Epg5 knockdown cells .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O

(K and L) EGFR degradation in control and Epg5 knockdown cells.


( M and N ) Distribution of Alexa Fluor 488 - conjugated transferrin at different time points in control siRNA - and Epg5 siRNA - treated cells . Higher magnification views are shown in the insets . Error bars indicate SEMs .

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

(M and N) Distribution of Alexa Fluor 488-conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs.


( A ) Co - immunoprecipitation ( Co - IP ) of Beclin1 with WT or mutant vBcl - 2 . 293T cells were transiently transfected with the indicated constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 and immunoblotting of V5 - tagged Beclin1 .

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

(A) Co-immunoprecipitation (Co-IP) of Beclin1 with WT or mutant vBcl-2. 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of V5-tagged Beclin1.


( B ) Co - IP of WT or mutant vBcl - 2 with endogenous Beclin1 . 293T cells were transfected with the indicated vBcl - 2 constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 and immunoblotting of endogenous Beclin1 . 1 % whole - cell lysates ( WCLs ) was used as the input .

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

(B) Co-IP of WT or mutant vBcl-2 with endogenous Beclin1. 293T cells were transfected with the indicated vBcl-2 constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of endogenous Beclin1. 1% whole-cell lysates (WCLs) was used as the input.


( C ) vBcl - 2 interaction with Bak protein . 293T cells were transfected with WT and mutant forms of vBcl - 2 as indicated . At 48 h posttransfection , WCLs were mixed either with GST - BakΔTM fusion protein ( left panel ) or with GST alone ( right panel ) for an in vitro GST pull - down ( GST PD ) assays . GST fusion proteins used for the pulldown assay are indicated ( bottom panel ) . 1 % WCL was used as the input . Data are representative of at least three experiments yielding similar results .

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

(C) vBcl-2 interaction with Bak protein. 293T cells were transfected with WT and mutant forms of vBcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (left panel) or with GST alone (right panel) for an in vitro GST pull-down (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results.


( A ) NIH3T3 cells stably expressing the WT and mutant forms of vBcl - 2 were transfected with GFP - LC3 . At 18 h posttransfection , cells were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as mean ± SEM of the combined results from three independent experiments . * * , P < 0 . 0001 .

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

(A) NIH3T3 cells stably expressing the WT and mutant forms of vBcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as mean±SEM of the combined results from three independent experiments. **, P<0.0001.


( B ) NIH3T3 cells expressing the WT or mutant forms of vBcl - 2 as indicated were transfected with GFP - LC3 and treated with 2 µM rapamycin for 6 h . GFP - LC3 was detected using an inverted fluorescence microscope ( top ) . Autophagy was quantified as mean ± SEM of the combined results from three independent experiments . Scale bar , 5 µm ; * * , P < 0 . 01 .

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

(B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01.


( C ) NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 , as indicated , were treated with 2 µM rapamycin . LC3 - I and LC3 - II levels were then determined by immunoblotting with an antibody against LC3 ( top ) . Densitometric quantification of the LC3 - II / LC3 - I ratios under normal and rapamycin treatment conditions is shown at the bottom . Similar results were obtained from three independent experiments .

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

(C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.


( top ) Representative confocal images of GFP - LC3 and HA - tagged vBcl - 2 ( WT and mutants ) in NIH3T3 cells infected with the indicated viruses ( MOI = 5 ) . Nuclei were counterstained with 4 ′ , 6 ′ - diamidino - 2 - phenylindole ( DAPI ) . Scale bar , 5 µm . ( bottom ) Quantification of the percentage of virally infected cells with GFP - LC3 punctate staining . Results shown represent mean ± SEM of combined results from three independent experiments ( 200 cells per experimental condition ) . * , P < 0 . 01 versus HA - WT ; * * , P < 0 . 001 versus HA - WT .

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

(top) Representative confocal images of GFP-LC3 and HA-tagged vBcl-2 (WT and mutants) in NIH3T3 cells infected with the indicated viruses (MOI = 5). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (bottom) Quantification of the percentage of virally infected cells with GFP-LC3 punctate staining. Results shown represent mean±SEM of combined results from three independent experiments (200 cells per experimental condition). *, P<0.01 versus HA-WT; **, P<0.001 versus HA-WT.


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) ,

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NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A),


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) , or for apoptosis by TUNEL staining ( B )

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

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B)


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) , or for apoptosis by TUNEL staining ( B ) or for the caspase 3 activation using flow cytometry ( C ) . Apoptotic cells in ( B ) were counted under high power magnification ( 60 × magnification ) . Mock , untreated condition . Data represents mean ± SEM of combined results from three independent experiments . Scale bars , 100 µm ( A ) , 5 µm ( B ) . * * , P < 0 . 0001 versus vector cells .

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

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.


( A ) Single - step ( right ) and multiple - step ( left ) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells .

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

(A) Single-step (right) and multiple-step (left) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells.


( B ) Acute replication of the WT and mutant vBcl - 2 γHV68 viruses in the lungs of BALB / c mice at 5 dpi ( up ) and 7 dpi ( down ) after intranasal infection determined by viral titers in the lungs of the infected mice ( left ) or by real - time PCR of the viral genomic DNA ( right ) . Mean ± SEM of five mice per group / experiment . Data of 7 dpi is pooled from two separate experiments . The vBcl - 2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [ for Δα1 , P = 0 . 82 ( day 5 ) ; P = 0 . 08 ( day 7 ) ; for ΔBH2 , P = 0 . 49 ( day 5 ) ; P = 0 . 54 ( day 7 ) ; unpaired t - tests ] and in viral genome loads in the lungs [ for Δα1 , P = 0 . 25 ( day 5 ) ; P = 0 . 28 ( day 7 ) ; for ΔBH2 , P = 0 . 21 ( day 5 ) ; P = 0 . 37 ( day 7 ) ; unpaired t - tests ] . n . s . , not significant .

O O O O O O O O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Acute replication of the WT and mutant vBcl-2 γHV68 viruses in the lungs of BALB/c mice at 5 dpi (up) and 7 dpi (down) after intranasal infection determined by viral titers in the lungs of the infected mice (left) or by real-time PCR of the viral genomic DNA (right). Mean±SEM of five mice per group/experiment. Data of 7 dpi is pooled from two separate experiments. The vBcl-2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [for Δα1, P = 0.82 (day 5); P = 0.08 (day 7); for ΔBH2, P = 0.49 (day 5); P = 0.54 (day 7); unpaired t-tests] and in viral genome loads in the lungs [for Δα1, P = 0.25 (day 5); P = 0.28 (day 7); for ΔBH2, P = 0.21 (day 5); P = 0.37 (day 7); unpaired t-tests]. n.s., not significant.


Splenic infectious centers measured at 12 dpi ( A , left ) , 14 dpi ( B , left ) , 21 dpi ( C , left ) , 28 dpi ( D , up ) , 35 dpi ( E , up ) , or 42 dpi ( F , up ) and viral genome load measured by real - time PCR at 12 dpi ( A , right ) , 14 dpi ( B , right ) , 21 dpi ( C , right ) , 28 dpi ( D , down ) , 35 dpi ( E , down ) , or 42 dpi ( F , down ) , in the BALB / cmice intranasally infected with the WT or recombinant γHV68 mutants , as indicated ( Mean ± SEM of five mice per group / time point / experiment ) . Data of 14 dpi and 28 dpi is pooled from two and three separate experiments , respectively . Preformed infectious virus was negligible in all spleen samples . No significant difference was detected at 12 dpi ( A ) , 14 dpi ( B ) , and 21 dpi ( C ) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [ for Δα1 , P = 0 . 58 ( day 12 ) ; P = 0 . 75 ( day 14 ) ; P = 0 . 18 ( day 21 ) ; for ΔBH2 , P = 0 . 64 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 81 ( day 21 ) ; unpaired t - tests ] and in viral genome loads [ for Δα1 , P = 0 . 85 ( day 12 ) ; P = 0 . 76 ( day 14 ) ; P = 0 . 96 ( day 21 ) ; for ΔBH2 , P = 0 . 56 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 25 ( day 21 ) ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased infectious center titers ( top ) of vBcl - 2 mutant viruses , as compared to WT γHV68 , were statistically significant [ vBcl - 2Δα1 versus vBcl - 2AAA ( 42 dpi ) , P = 0 . 46 ; vBcl - 2Δα1 versus WT γHV68 ( 42 dpi ) , P = 0 . 08 ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased viral DNA loads ( bottom ) of the vBcl - 2Δα1 , vBcl - 2AAA , and vBcl - 2 null mutants , as compared to WT γHV68 and the vBcl - 2ΔBH2 mutant , were statistically significant as follows ( unpaired t - tests ) : at day 28 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 01 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 01 ; vBcl - 2 - null versus WT γHV68 , P < 0 . 01 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2 - null versus vBcl - 2ΔBH2 , P < 0 . 001 ; at day 35 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 05 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 05 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 01 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 05 ; at day 42 , vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2ΔBH2 versus WT γHV68 , P = 0 . 33 . I . C . , infectious center . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/cmice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.


( A ) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases . The x - axis contains the mutation coefficients ( effect sizes ) and the y - axis the adjusted P - values . The vertical line in the x - axis is at x = 0 and separates the positive ( right ) from the negative ( left ) associations . The horizontal lines in the y - axis represent the points of statistical significance in the plot ( adjusted P - values < 0 . 05 and < 0 . 01 ) . The associations are represented in the form of a mutated gene - kinase . The colour gradient represents the string network edge weight interval of the pair ( grey if the pair is not in the string network ) . ( B ) Same as ( A ) for the TFs . The inner plot shows the effects of TF mutations on their own activities . (

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases. The x-axis contains the mutation coefficients (effect sizes) and the y-axis the adjusted P-values. The vertical line in the x-axis is at x=0 and separates the positive (right) from the negative (left) associations. The horizontal lines in the y-axis represent the points of statistical significance in the plot (adjusted P-values < 0.05 and < 0.01).The associations are represented in the form of a mutated gene - kinase. The colour gradient represents the string network edge weight interval of the pair (grey if the pair is not in the string network). (B) Same as (A) for the TFs. The inner plot shows the effects of TF mutations on their own activities. (


( C ) ( D ) Examples of the genetic associations highlighted in the volcano plots . The x - axis represents the associations and the y - axis the protein activities . The colours stratify the samples by their mutational status in the respective genes . The outliers ( defined as the data points beyond Q1 - 1 . 5 * IQR and Q3 + 1 . 5 * IQR , where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range ) were removed from the distributions for representation purposes . The number of protein activity quantifications ( including outliers ) are shown beneath each boxplot . The P - values from Wilcoxon rank sum tests comparing both distributions are shown . All data points ( including outliers ) were used to calculate the P - values .

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

(C) (D) Examples of the genetic associations highlighted in the volcano plots. The x-axis represents the associations and the y-axis the protein activities. The colours stratify the samples by their mutational status in the respective genes. The outliers (defined as the data points beyond Q1-1.5*IQR and Q3+1.5*IQR, where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range) were removed from the distributions for representation purposes. The number of protein activity quantifications (including outliers) are shown beneath each boxplot. The P-values from Wilcoxon rank sum tests comparing both distributions are shown. All data points (including outliers) were used to calculate the P-values.


( C ) Kinase activity gradient along the samples for a selection of the kinases shown in ( B ) .

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

(C) Kinase activity gradient along the samples for a selection of the kinases shown in (B).


( F ) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples . The trend line and the Pearson ' s r , with the respective P - value , are shown . In red and green are the kinases preferably regulated in the tumours and in the conditions , respectively . In blue are the kinases regulated in both . The 95 % confidence interval around the regression line is represented by the shaded area .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples. The trend line and the Pearson's r, with the respective P-value, are shown. In red and green are the kinases preferably regulated in the tumours and in the conditions, respectively. In blue are the kinases regulated in both. The 95% confidence interval around the regression line is represented by the shaded area.


( C - F ) Kaplan - Meier survival plots comparing the survival probabilities ( y - axes ) as a function of time in days ( x - axes ) for proteins and cancer types shown in ( A ) and ( B ) . ( C ) MYC / brain cancer ( inactive = 71 , neutral = 39 , active = 67 ) . ( D ) MYC / liver cancer ( inactive = 58 , neutral = 59 , active = 40 ) . ( E ) MAP3K8 / renal cancer ( neutral = 95 , active = 7 ) . ( F ) PRKCA / renal cancer ( inactive = 3 , neutral = 92 , active = 7 ) . The log - rank P - values are shown in the plots .

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

(C-F) Kaplan-Meier survival plots comparing the survival probabilities (y-axes) as a function of time in days (x-axes) for proteins and cancer types shown in (A) and (B). (C) MYC/brain cancer (inactive = 71, neutral = 39, active = 67). (D) MYC/liver cancer (inactive = 58, neutral = 59, active = 40). (E) MAP3K8/renal cancer (neutral = 95, active = 7). (F) PRKCA/renal cancer (inactive = 3, neutral = 92, active = 7). The log-rank P-values are shown in the plots.


( c ) Shown are the responses of the glucose sensor promoter ( PgluA7 ) , a negative control lacking the CRP operator ( PgluA7 * ) , and a constitutive promoter ( BBa _ J23101 ) to the presence ( + ) and absence ( - ) of

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

(c) Shown are the responses of the glucose sensor promoter (PgluA7), a negative control lacking the CRP operator (PgluA7*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of glucose