Figure 4. PAX4 induces proteasome genes... | Rockefeller University Press

Figure 4.

$PAX4 induces proteasome genes in vivo. (A) Proteasome gene expression was measured by RT-PCR analysis of mRNA preparations from innervated and 10-day denervated muscles expressing shPAX4 or shLacz control. Means ± SEM are presented as a ratio to innervated. N = 5 mice per condition. *, P < 0.05, **, P < 0.005, ***, P < 0.0005 versus innervated shLacz; #, P < 0.05 and ##, P < 0.005 versus denervated shLacz by one-tailed unpaired Student’s t test. (B) PAX4 downregulation with shPAX4 reduces fiber atrophy on denervation. Measurement of cross-sectional areas of 1,178 fibers from 10-day denervated fibers expressing shPAX4 (green fibers, also express GFP) vs. 1,178 adjacent non-transfected fibers. N = 3 mice. Statistics in Table 1. Right: representative image of transfected muscle. Fiber membrane staining (red) using WGA. Scale bar: 50 μm. (C) Top: Strategy for generating PAX4 conditional KO mice. Middle: DNA was isolated from tail snippings and analyzed by PCR genotyping. Following tamoxifen injections of Cre+/−/PAX4fl/fl mice, removal of PAX4 from the genome was verified using the P1/P3 primers (Table S3). Bottom: nuclear extracts from WT and KO mice were analyzed by immunoblotting using PAX4 antibody. (D) Body weight (g) and survival (%) of PAX4 KO and WT mice are presented, following tamoxifen injections and muscle denervation. (E and F) PAX4 mRNA levels increase on denervation of WT mouse muscle but are absent in muscles from PAX4 KO mice. Mice were injected with tamoxifen and their muscles denervated. PCR (E) or RT-PCR (F) using primers for PAX4 was performed on mRNA preparations from innervated and denervated muscles. Means ± SEM are presented as a ratio to WT innervated. N = 5 mice per condition. *, P < 0.05 versus innervated in WT; #, P < 0.05 versus denervated in WT by one-tailed unpaired Student’s t test. (G and H) Proteasome gene expression was measured by RT-PCR analysis of mRNA preparations from innervated and 10 days (G) or 3 days (H) denervated muscles from WT and PAX4 KO mice. Means ± SEM are presented as ratio to WT innervated. N = 5 mice per condition. *, P < 0.05 and **P < 0.005 vs. innervated in WT; #, P < 0.05 and ##, P < 0.005 versus denervated in WT by one-tailed unpaired Student’s t test. (I) PAX4 enters the nucleus at 3 days after denervation. Cytosolic and the corresponding nuclear fractions from innervated and denervated (at 3, 7, 10 days) muscles were analyzed by SDS-PAGE and immunoblotting. (J) PAX4 binds the promoter region of PSMC2 gene. ChIP was performed on innervated and denervated (10 days) muscles from WT or PAX4 KO mice using PAX4 antibody or non-specific IgG control, and primers for the PSMC2 gene. Data are plotted as mean fold change relative to IgG control ± SEM and represents three independent experiments. N = 3 mice per condition. *, P < 0.05 versus IgG in WT; #P < 0.05 versus denervated in WT by one-tailed unpaired Student’s t test. (K) Correlative reduction in protein levels was confirmed for representative genes by analysis of soluble fractions from innervated and denervated muscles from WT or PAX4 KO mice by SDS-PAGE and immunoblotting. (L) The content of active assembled proteasomes increase in denervated muscles, but not in muscles lacking PAX4. Measurement of proteasome content by native gels and immunoblotting and proteasome peptidase activity by LLVY-AMC hydrolysis. Source data are available for this figure: SourceData F4.$

PAX4 induces proteasome genes in vivo. (A) Proteasome gene expression was measured by RT-PCR analysis of mRNA preparations from innervated and 10-day denervated muscles expressing shPAX4 or shLacz control. Means ± SEM are presented as a ratio to innervated. N = 5 mice per condition. *, P < 0.05, **, P < 0.005, ***, P < 0.0005 versus innervated shLacz; #, P < 0.05 and ##, P < 0.005 versus denervated shLacz by one-tailed unpaired Student’s t test. (B) PAX4 downregulation with shPAX4 reduces fiber atrophy on denervation. Measurement of cross-sectional areas of 1,178 fibers from 10-day denervated fibers expressing shPAX4 (green fibers, also express GFP) vs. 1,178 adjacent non-transfected fibers. N = 3 mice. Statistics in Table 1. Right: representative image of transfected muscle. Fiber membrane staining (red) using WGA. Scale bar: 50 μm. (C) Top: Strategy for generating PAX4 conditional KO mice. Middle: DNA was isolated from tail snippings and analyzed by PCR genotyping. Following tamoxifen injections of Cre^+/−/PAX4^fl/fl mice, removal of PAX4 from the genome was verified using the P1/P3 primers (Table S3). Bottom: nuclear extracts from WT and KO mice were analyzed by immunoblotting using PAX4 antibody. (D) Body weight (g) and survival (%) of PAX4 KO and WT mice are presented, following tamoxifen injections and muscle denervation. (E and F) PAX4 mRNA levels increase on denervation of WT mouse muscle but are absent in muscles from PAX4 KO mice. Mice were injected with tamoxifen and their muscles denervated. PCR (E) or RT-PCR (F) using primers for PAX4 was performed on mRNA preparations from innervated and denervated muscles. Means ± SEM are presented as a ratio to WT innervated. N = 5 mice per condition. *, P < 0.05 versus innervated in WT; #, P < 0.05 versus denervated in WT by one-tailed unpaired Student’s t test. (G and H) Proteasome gene expression was measured by RT-PCR analysis of mRNA preparations from innervated and 10 days (G) or 3 days (H) denervated muscles from WT and PAX4 KO mice. Means ± SEM are presented as ratio to WT innervated. N = 5 mice per condition. *, P < 0.05 and **P < 0.005 vs. innervated in WT; #, P < 0.05 and ##, P < 0.005 versus denervated in WT by one-tailed unpaired Student’s t test. (I) PAX4 enters the nucleus at 3 days after denervation. Cytosolic and the corresponding nuclear fractions from innervated and denervated (at 3, 7, 10 days) muscles were analyzed by SDS-PAGE and immunoblotting. (J) PAX4 binds the promoter region of PSMC2 gene. ChIP was performed on innervated and denervated (10 days) muscles from WT or PAX4 KO mice using PAX4 antibody or non-specific IgG control, and primers for the PSMC2 gene. Data are plotted as mean fold change relative to IgG control ± SEM and represents three independent experiments. N = 3 mice per condition. *, P < 0.05 versus IgG in WT; #P < 0.05 versus denervated in WT by one-tailed unpaired Student’s t test. (K) Correlative reduction in protein levels was confirmed for representative genes by analysis of soluble fractions from innervated and denervated muscles from WT or PAX4 KO mice by SDS-PAGE and immunoblotting. (L) The content of active assembled proteasomes increase in denervated muscles, but not in muscles lacking PAX4. Measurement of proteasome content by native gels and immunoblotting and proteasome peptidase activity by LLVY-AMC hydrolysis. Source data are available for this figure: SourceData F4.