Journal: bioRxiv

Article Title: Meteorin-like (Metrnl) adipomyokine improves glucose tolerance in type 2 diabetes via AMPK pathway

doi: 10.1101/420489

Figure Lengend Snippet: Metrnl increases GLUT4 expression by stimulating HDAC5 phosphorylation. a . Total mRNA from C2C12 cells were prepared after metrnl (100 ng/mL) treatment for the indicated times, and real-time qRT-PCR was performed using GLUT4-specific primers. For these assays, β-actin mRNA served as a positive control. b . C2C12 cells were stimulated with metrnl (100 ng/mL) for the indicate4d times. The cell lysates were analyzed by Western blotting using antibodies against GLUT4, with β-actin as a control. c . Time-dependent phosphorylation of HDAC5 after metrnl treatment. C2C12 cells were incubated with metrnl (100 ng/mL) for the indicated times. Cell lysates were analyzed by Western blotting usingantibodies against phospho-HDAC5 (Thr 498 ), while HDAC5 served as controls. d . C2C12 cells were pre-treated with compound C (30 μM), then treated with metrnl (100 ng/mL). Cell lysates were analyzed by Western blotting using an antibody against phospho-HDAC5 (Thr 498 ); HDAC5 served as a control. e . C2C12 cells were transiently transfected with AMPKα2 siRNA or non-target siRNA. Cell lysates were analyzed by Western blotting using antibodies against phospho-HDAC5 (Thr 498 ) and AMPKα2; HDAC5 and β-actin served as controls. f . C2C12 cells were treated with metrnl (100 ng/mL). Cytosolic and nuclear proteins were extracted from the cells. The phosphorylation of HDAC5 was evaluated by Western blot analysis. HDAC5 served as a control. Western blotting was performed on nuclear and cytosolic fractions to detect nuclear (lamin B) and cytosolic (α-tubulin) marker proteins. g . Representative images of phospho-HDAC5 treated with metrnl for 30?min. Scale bars, 10?μm. h . C2C12 cells were immunoprecipitated with anti-14-3-3 antibody, followed by Western blotting using anti-phospho-HDAC5. i . Representative images (phospho-HDAC5 and 14-3-3 objective images) of cells treated with metrnl for 1?h. Scale bars, 10?μm. j . The relative occupancy of HDAC5 and AcH3 on the GLUT4 promoter was assessed by qChIP analyses following 60 min of metrnl (100 ng/mL) treatment. The ChIP data represent IP values for each region’s ratio relative to the input. The results shown are from three independent experiments. * P < 0.05 and ** P < 0.01 vs. control, as indicated. Results are displayed as the mean ± SD from three experiments.

Article Snippet: HDAC5 was immunoprecipitated using an HDAC5 antibody (Novus Biologicals; Littleton, CO, USA), or an unrelated antibody (normal rabbit IgG) for a control.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Incubation, Transfection, Marker, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic tumor images and tumor weights from KPC Hdac5-WT and KPC Hdac5-KO mouse models treated with vehicle or MRTX1133 (30 mg/kg, i.p., twice daily [bid]) ( n = 5). ( B ) Kaplan-Meier survival curves with log-rank test ( n = 5). ( C and D ) Representative IHC images of tumors in C . IHC scores were quantified in D . Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. ( E ) C57BL/6 mice were orthotopically injected with KPC-Luc cells expressing shNc or shHdac5. Bioluminescence imaging was performed on day 7, followed by treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid). Representative bioluminescence images and corresponding quantification were acquired on day 28 ( n = 5). ( F ) Tumor growth curves in PDX models with treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid) ( n = 5). ( G and H ) Representative IHC images of PDXs and quantified IHC scores ( H ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( A , D – F , and H ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Injection, Expressing, Imaging

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of genes upregulated in KPC Hdac5-KO mice ( n = 5 per genotype; log 2 [fold-change] > 1, P < 0.05, 2,410 genes) identified via RNA-Seq and genes upregulated in shHDAC5-treated PANC-1 cells ( n = 3 per condition; log 2 [fold-change] > 1, P < 0.05, 2,683 genes), revealing a shared subset of 378 genes. ( B ) Bar graph showing the top 10 enriched transcription factors from transcription factor analysis of 378 genes in A using Enrichr. ATF2, activating transcription factor 2. ( C ) Dual-luciferase reporter assays were performed to assess the transcriptional activity of c-Myc in HDAC5-depleted PANC-1 and AsPC-1 cells. Data are presented as mean ± SD ( n = 5). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. ( D ) Heatmap of MYC ChIP-Seq signal intensity (±3 kb around MYC binding sites) in control vs. HDAC5-knockdown PANC-1 cells. ( E ) The average ChIP signal of MYC centered at transcription start site (±3 kb) in indicated groups. ( F ) Venn diagram depicting the overlap between genes with enhanced MYC promoter occupancy after shHDAC5 knockdown, as identified by ChIP-Seq, and genes upregulated in PANC-1 cells following shHDAC5 knockdown, as determined by RNA-Seq. P = 6 × 10 –71 . ( G ) KEGG pathway enrichment analysis of 821 intersecting genes in F . ( H and I ) Western blot analysis of canonical MAPK pathway protein expression under indicated conditions.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: RNA Sequencing, Luciferase, Activity Assay, ChIP-sequencing, Binding Assay, Control, Knockdown, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic images of pancreatic tumors from KPC Hdac5-WT and KPC Hdac5-KO mice after sacrifice. ( B ) Tumor weight analysis in KPC mice treated with vehicle, MRTX1133 (30 mg/kg, i.p. bid), MYCi975 (50 mg/kg, i.p. bid), or their combination ( n = 5). ( C ) Kaplan-Meier survival curves with log-rank test ( n = 5). *** P < 0.001. ( D and E ) Representative IHC images of tumors from treated mice and quantified IHC scores ( E ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent quantifications. ( F and G ) Representative bioluminescence images and quantification ( n = 5). ( H ) Macroscopic images of PDX tumors after 30 days of treatment. ( I and J ) Tumor growth curves ( I ) and final tumor weights ( J ) in PDX models treated with Vehicle + LV-Control, MRTX1133 (30 mg/kg, i.p. bid) + LV-Control, Vehicle + LV-sgMYC (50 μL lentivirus, s.c., weekly), or MRTX1133 + LV-sgMYC. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( B , E , G , I , and J ). *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Control