Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of CDK8 gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Genome Wide, CRISPR, shRNA, In Vitro, Cell Counting, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Correlation between CDK8 gene dependency and sensitivity to the CDK8 inhibitor BI-1347 in 669 cancer cell lines (y-axis showing CDK8 dependency score; x-axis showing PRISM LFC value of BI-1347 treatment). Linear regression lines and Pearson correlation coefficients (R) are shown for all cell lines (black), aRMS (red), and eRMS (blue). B. Western blot analysis showing CDK8 inhibition by small molecules as assessed by STAT1 phosphorylation at serine 727. C, D. Dose response curves of inactive BI-1347 analog (BI-1374) (C) and three pharmacologic CDK8 inhibitors (D), BI-1347, SEL-120-34A, and JH-XII-178, at day 7 of treatment in Rh30, Rh4 and RHJT cell lines. E, F. Live cell proliferation assessed by Incucyte for Rh30 (E) and Rh4 (F) cells after treatment with vehicle DMSO (black), the CDK8 inhibitor BI-1347 (red) and its inactive analog BI-1374 (gray). Data were normalized to DMSO. Data represent means ± SEM (n=6, *: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. Line graph reveals mean subcutaneous tumor volume (mm ) formed by Rh30 cells after treatment with the CDK8 inhibitor SEL-120-34A. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Western Blot, Inhibition, Phospho-proteomics

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plots showing the number of gene body changes after 24 hrs of CDK8 inhibitor BI-1347 treatment. Significantly up-regulated genes are highlighted in red; significantly downregulated genes are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). B. Metagene plots of PRO-seq reads of genes with significant upregulation or downregulation of gene body transcription at 24 hrs of BI-1347 treatment. C. Heatmap of Log 2 transformed fold change values of PRO-seq pausing indices of genes with changes in gene body transcription at the 24 hr time point. D. Heatmap showing log 2 transformed fold change of RNA-seq read counts for indicated gene sets after DMSO and BI-1347 (10 nM) treatment for 24 or 72 hrs. E. Bubble dot plot shows Gene Set Enrichment Analysis (GSEA) of PRO-seq gene hits that are upregulated at 24 hrs. Dot size indicates number of genes in each gene set, and dot color indicates p -adjusted value. F. Immunofluorescence analysis of myogenin (green; 20X) in Rh30 (left) and Rh4 (right) cells after 7 days of DMSO or BI-1347 treatment. G. Representative images of H&E stain of xenograft tumors. Arrows indicate myofibrils. H. Volcano plots reveal the number of changes in enhancer RNA (eRNA) transcripts after 24 hrs of BI-1347 treatment. Significantly upregulated eRNAs are highlighted in red; significantly downregulated eRNAs are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5) I. MA plots showing the changes of chromatin accessibility by ATAC-seq following 4 and 24 hrs of BI-1347 treatment. Significantly increased ATAC-seq peaks are highlighted in red; significantly decreased ATAC-seq peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). J. Pie chart showing the peak annotation of up-regulated ATAC-seq peaks to gene features at 24 hrs of BI-1347 treatment. K. Histogram of PRO-seq reads around up-regulated ATAC-seq peaks at indicated time points of BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Transformation Assay, RNA Sequencing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. IGV gene tracks showing a time course analysis of BI-1347 treatment by PRO-seq signal at the PAX3::FOXO1 locus. B. Heatmaps of CUT&RUN analysis of CDK8 and PAX3::FOXO1 binding in Rh30 and Rh4 cell lines. C. MA plots showing changes of PAX3::FOXO1 binding following BI-1347 treatment at 24 and 72 hrs. Significantly increased PAX3::FOXO1 peaks are highlighted in red; significantly decreased PAX3::FOXO1 peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). D, E. Heatmaps of CDK8 (D) and PAX3::FOXO1 (E) signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. F. Heatmaps of log 2 transformed fold change of RNA-seq read counts plotted by the shared gene between 24 hrs of PAX3::FOXO1 degradation and 72 hrs of CDK8 inhibition at the indicated time points. G. IGV gene tracks showing a time course analysis for PRO-seq signal at the VGLL2 locus after BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Binding Assay, Transformation Assay, RNA Sequencing, Inhibition

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Scatter plot showing the Z-scored average log2 fold change (LFC) of gene knockout effects in Rh30 cells treated with BI-1347 versus DMSO at day 14 (y-axis) and day 21 (x-axis) from genome-wide CRISPR-Cas9 screens. Each point represents an individual gene; dot size corresponds to statistical significance, and dot color indicates classification. B. Bubble dot plot of GSEA for gene hits scoring at day 21 in the CRISPR-Cas9 BI-1347 drug modifier screen ranked by C5 gene sets. C. Box plots showing construct-level Z-score averages for individual genes in the SAGA complex from the genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by SAGA functional modules. D-F. Live cell proliferation assessed by Incucyte for BI-1347+/-sgTADA2B ( D ), BI-1347+/-sgTAF5L ( E ), and BI-1347+/-GSK699 ( F ). G. Quantitative real-time TaqMan qPCR analysis of RUNX1 , SEMA3D , and VGLL 2 expression at day 7 following treatment with vehicle control (DMSO), CDK8 inhibitors, or GSK699, and the indicated combination treatments. Expression levels were normalized to GAPDH gene expression and shown relative to DMSO control. Data represent means ± SEM (n=6).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Gene Knockout, Genome Wide, CRISPR, Construct, Functional Assay, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Live cell proliferation assay by Incucyte for BI-1347 treatment combined with CRISPR knockout of SIX4 with two different guide RNAs. B. Western blot analysis of SIX4 protein level after treatment with three CDK8 inhibitors at indicated time points. Lamin B included as a loading control. C, D. Western blot analysis of SIX4 protein levels from cytoplasmic and nuclear fractions from Rh30 ( C ) and Rh4 ( D ) cells treated with DMSO or BI-1347 for 14 days. GAPDH and Lamin B served as nuclear and cytoplasmic loading controls, respectively. E, F. Volcano showing log 2 fold change of SIX4 ( E ) and TADA2B ( F ) genome binding sites determined by CUT&RUN analysis. Red highlights significantly up regulated sites and blue highlights significantly down regulated sites (log 2 FC 1.5, padj<0.05). G, H. Pie charts showing the annotation of upregulated CUT&RUN peaks for SIX4 ( G ) and TADA2B ( H ) sites after 24 hrs of BI-1347 treatment in Rh30 cells. I. Heatmaps of time course analysis of ATAC-seq signal, SIX4 signal, TADA2B signal, H3K27ac signal around upregulated ATAC-seq peaks at 24 hrs of BI-1347 treatment. J. IGV gene tracks showing the PRO-seq, SIX4, TADA2B, ATAC-seq, PAX3::FOXO1, CDK8, H3K4me3, and H3K27ac at the VGLL2 gene body and enhancers loci at indicated time points of BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Proliferation Assay, CRISPR, Knock-Out, Western Blot, Control, Binding Assay

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

doi: 10.1083/jcb.201008167

Figure Lengend Snippet: SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A–C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1. (D–F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3 vac ; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Unspecific binding sites were saturated by incubating membranes for 1 h in 0.05% Tween 20 (vol/vol in TBS) supplemented with 5% nonfat powdered milk (wt/vol in TBS) followed by overnight incubation with primary antibodies specific for acetylated lysine, phosphoacetyl-CoA carboxylase α (Ser79), acetyl-CoA carboxylase α, phospho-AKT1 (Thr308), AKT1, LC3, phosphomitogen-activated protein kinase 8 (Tyr183/Thr185), phospho-PRKAA1 (Thr172), PRKAA1, cyclin-dependent kinase inhibitor 1B, phosphoprotein tyrosine kinase 2 β (Tyr402), phosphoribosomal protein S6 kinase (Thr421/Ser424), ribosomal protein S6 kinase, stress-induced phosphoprotein 1 (Cell Signaling Technology), ATG7 (Sigma-Aldrich), phosphocyclin-dependent kinase inhibitor 1B (Abcam), TP53 (DO7), p62, SIRT1 (Santa Cruz Biotechnology), and peptidylprolyl isomerase A (cyclophilin A; Enzo Life Sciences, Inc.).

Techniques: Activity Assay, Cell Culture, Control, Transfection, Plasmid Preparation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

doi: 10.1083/jcb.201008167

Figure Lengend Snippet: Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A–C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown. (B) Clustering analysis for the effects on protein kinase phosphorylation. (C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data. (D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3 vac ). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Unspecific binding sites were saturated by incubating membranes for 1 h in 0.05% Tween 20 (vol/vol in TBS) supplemented with 5% nonfat powdered milk (wt/vol in TBS) followed by overnight incubation with primary antibodies specific for acetylated lysine, phosphoacetyl-CoA carboxylase α (Ser79), acetyl-CoA carboxylase α, phospho-AKT1 (Thr308), AKT1, LC3, phosphomitogen-activated protein kinase 8 (Tyr183/Thr185), phospho-PRKAA1 (Thr172), PRKAA1, cyclin-dependent kinase inhibitor 1B, phosphoprotein tyrosine kinase 2 β (Tyr402), phosphoribosomal protein S6 kinase (Thr421/Ser424), ribosomal protein S6 kinase, stress-induced phosphoprotein 1 (Cell Signaling Technology), ATG7 (Sigma-Aldrich), phosphocyclin-dependent kinase inhibitor 1B (Abcam), TP53 (DO7), p62, SIRT1 (Santa Cruz Biotechnology), and peptidylprolyl isomerase A (cyclophilin A; Enzo Life Sciences, Inc.).

Techniques: Control, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

doi: 10.1083/jcb.201008167

Figure Lengend Snippet: Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3–transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3 vac ). (C–E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity. (D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3 vac ). (B and D) Means ± SEM; n = 3; *, P < 0.05. (E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Unspecific binding sites were saturated by incubating membranes for 1 h in 0.05% Tween 20 (vol/vol in TBS) supplemented with 5% nonfat powdered milk (wt/vol in TBS) followed by overnight incubation with primary antibodies specific for acetylated lysine, phosphoacetyl-CoA carboxylase α (Ser79), acetyl-CoA carboxylase α, phospho-AKT1 (Thr308), AKT1, LC3, phosphomitogen-activated protein kinase 8 (Tyr183/Thr185), phospho-PRKAA1 (Thr172), PRKAA1, cyclin-dependent kinase inhibitor 1B, phosphoprotein tyrosine kinase 2 β (Tyr402), phosphoribosomal protein S6 kinase (Thr421/Ser424), ribosomal protein S6 kinase, stress-induced phosphoprotein 1 (Cell Signaling Technology), ATG7 (Sigma-Aldrich), phosphocyclin-dependent kinase inhibitor 1B (Abcam), TP53 (DO7), p62, SIRT1 (Santa Cruz Biotechnology), and peptidylprolyl isomerase A (cyclophilin A; Enzo Life Sciences, Inc.).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Control, Positive Control, Activity Assay, Expressing, Variant Assay, Mutagenesis, Fluorescence, Microscopy, Western Blot

Journal: The Journal of Cell Biology

Article Title: Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

doi: 10.1083/jcb.201008167

Figure Lengend Snippet: Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3 vac ). (bottom) Representative immunoblots showing endogenous LC3 lipidation. (B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C–E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3 vac . (C) Representative images. (D) Quantitative results. Bars represent the percentages (means ± SEM; n = 3; *, P < 0.05; and **, P < 0.01 as compared with the same tissue from untreated animals) of cells exhibiting GFP-LC3 vac . (E) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL/6 mouse tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Unspecific binding sites were saturated by incubating membranes for 1 h in 0.05% Tween 20 (vol/vol in TBS) supplemented with 5% nonfat powdered milk (wt/vol in TBS) followed by overnight incubation with primary antibodies specific for acetylated lysine, phosphoacetyl-CoA carboxylase α (Ser79), acetyl-CoA carboxylase α, phospho-AKT1 (Thr308), AKT1, LC3, phosphomitogen-activated protein kinase 8 (Tyr183/Thr185), phospho-PRKAA1 (Thr172), PRKAA1, cyclin-dependent kinase inhibitor 1B, phosphoprotein tyrosine kinase 2 β (Tyr402), phosphoribosomal protein S6 kinase (Thr421/Ser424), ribosomal protein S6 kinase, stress-induced phosphoprotein 1 (Cell Signaling Technology), ATG7 (Sigma-Aldrich), phosphocyclin-dependent kinase inhibitor 1B (Abcam), TP53 (DO7), p62, SIRT1 (Santa Cruz Biotechnology), and peptidylprolyl isomerase A (cyclophilin A; Enzo Life Sciences, Inc.).

Techniques: In Vitro, In Vivo, Transfection, Plasmid Preparation, Cell Culture, Control, Western Blot, Transgenic Assay, Expressing, Injection, Immunofluorescence, Microscopy