Journal: The Journal of Clinical Investigation

Article Title: HDAC inhibitors elicit metabolic reprogramming by targeting super-enhancers in glioblastoma models

doi: 10.1172/JCI129049

Figure Lengend Snippet: (A) GBM cells were treated with Pb or Ro or chronically exposed to Pb or Ro (n = 3–4). (B) PCE analyses of U87 cells transfected with Myc siRNA and treated with Pb or Ro for 24 hours. (C) PCE analyses of U87 cells transfected with siRNA HDAC1, HDAC2, or a combination of both. (D and E) Real-time PCR analysis of U87 cells transfected with HDAC1 siRNA, HDAC2 siRNA, or a combination of both (n = 3–4). (F) ChIP-Seq profile of parental U87 and LN229 cells or U87 and LN229 cells chronically exposed to Pb with an antibody against H3K27ac or Rpb1. Shown are the respective tracks around the desert of the PPARGC1A (PGC1α) locus. (G) ChIP-qPCR (with anti-HDAC2 antibody) of the PGC1α promoter (c-Myc–binding region) from the indicated cell lysates (n = 3). (H) ChIP-qPCR of the PGC1α promoter (c-Myc–binding region) from the indicated cell lysates with either anti–c-Myc antibody or anti-H3K27ac antibody (n = 3). (I) PCE analysis of U87 cells transduced with a c-Myc construct and treated with 2.5 nM Ro for 24 hours. (J) Mitochondrial stress test of parental U87 cells or U87 cells chronically exposed to Pb and transduced with an shRNA against PGC1α (n = 4–5). O, oligomycin; F, FCCP; R/A, rotenone and antimycin A. (K) Maximal respiration data from the experiment in J. (L) Mitochondrial stress extracellular flux analysis of parental U87 cells or U87 cells chronically exposed to Pb and transduced with PGC1α sgRNAs (n = 4). (M) Maximal respiration data from the experiment in L. U87-KO-NT, nontargeting KO U87 cells; U87PbR-KO-NT, nontargeting KO U87 cells chronically exposed to Pb; U87-KO-PGC1A-2, PGC1A-2–KO U87 cells; U87PbR-KO-PGC1A-2, PGC1A-2–KO U87 cells chronically exposed to Pb. (N) PCE analysis of U87 cells transduced with an shRNA against PGC1α or PGC1α sgRNAs. Data represent the mean ± SD. Statistical significance was determined by 2-tailed Student’s t test (A) or 1-way ANOVA (D, E, G, H, K, and M). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following shRNAs were purchased from Santa Cruz Biotechnology: PGC1α (sc-38884-V), AMPKA (sc-29673-V), and PPARD (sc-36305-V).

Techniques: Transfection, Real-time Polymerase Chain Reaction, ChIP-sequencing, ChIP-qPCR, Binding Assay, Transduction, Construct, shRNA

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway.

doi: 10.1096/fj.202000492R

Figure Lengend Snippet: FIGURE 1 LRPGC1 translocates into the nucleus following LA stimulation. A, Schematic structures of PGC1α and LRPGC1. AD, activation domain; RD, repression domain; NES, nuclear export signal; NLS, putative nuclear localization signal; Ub, ubiquitination. B, RT-PCR of Lrpgc1, Pgc1α, and Gapdh in indicated rat tissues. Data are from two rats. Upper, primer design. C, Western blotting on whole cell extracts of COS-1 cells expressing PGC1α or LRPGC1 incubated with MG132 (0-5 μM) for 6 hours. Anti-PGC1α antibody which recognizes N-terminus of PGC1 proteins was used. GAPDH was detected as loading control. Experiments were repeated three times with similar results. D, Confocal live images of CFP- PGC1α or CFP-LRPGC1 expressed in COS-1 cells. Bar = 20 μm. E, F, Time-lapse confocal imaging of COS-1 cells expressing CFP-LRPGC1. Notably, LRPGC1 translocated from the cytoplasm to the nucleus after treatment with lactic acid (LA) (final conc. 7.5 mM). Experiments were repeated more than 10 times with similar results. Bars = 50 (E) or 20 (F) μm. G, Western blotting on liver nuclear extract of the rats before and after injection of 2 g/kg body weight of LA (15, 30, 60 minutes). LRPGC1 was detected by anti-PGC1α antibody as described above. Anti-lamin B1 antibody was used as loading control. Experiments were repeated twice with similar results. Left, time-course schema

Article Snippet: To generate a PGC1 KO cell line, HepG2 cells were transfected with PGC1α CRISPR/Cas9 KO Plasmid (h) (sc-400070; Santa Cruz Biotechnology) and PGC1α HDR Plasmid (h) (sc400070-HDR; Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen).

Techniques: Activation Assay, Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Incubation, Control, Imaging, Injection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway.

doi: 10.1096/fj.202000492R

Figure Lengend Snippet: FIGURE 3 LRPGC1 increases LA consumption through enhancement of ERRγ-mediated transcription. A, B, Transcription assay. COS-1 cells were transfected with pcDNA3.1-empty, pcDNA3.1-ERR (α, β, or γ), pcDNA3.1-LRPGC1, and ERRE-driven luciferase reporter construct (ERRE-Luc) and incubated with or without lactic acid (LA), as indicated. An actin promoter-driven β-galactosidase expression construct was transfected as an internal control. Luciferase activity was normalized by β-galactosidase activity (n = 4 per group). C, Time-lapse imaging of COS- 1 cells expressing CFP-LRPGC1 and YFP-ERRγ before and after treatment with LA (final conc. 7.5 mM). Bar = 20 μm. D-F, FRET microscopy on COS-1 cells expressing the indicated proteins. The cells were incubated with or without LA for 1 hour and fixed with 4% paraformaldehyde in 0.1M phosphate buffer for 10 minutes before FRET microscopy was performed. Bar = 10 μm. D, Ratio of YFP/CFP fluorescence intensity in ROIs under excitation at 458 nm (n = 34 for CFP-LRPGC1 + YFP-ERRγ, LA (−) and CFP-LRPGC1 + YFP, LA (+); n = 33 for CFP- LRPGC1 + YFP-ERRγ, LA (+)). E, Acceptor photobleaching. Increase of donor (CFP-LRPGC1, at 473 nm) fluorescence intensity was calculated after photobleaching of ROIs (YFP-ERRγ, at 514 nm) (n = 34 for CFP-LRPGC1 + YFP-ERRγ, LA (−) and CFP-LRPGC1 + YFP, LA (+); n = 33 for CFP-LRPGC1 + YFP-ERRγ, LA (+)). F, Pre- and post-bleached pseudocolor images. White circles denote the bleached ROI. Magnified ROI images are shown on the Right. G-I, Comparison of LA consumption between HepG2 and PGC1 KO cells (G), and among KO cells transfected with pcDNA3.1-empty, pcDNA3.1-PGC1α, or pcDNA3.1-LRPGC1 (H), or with pcDNA3.1-empty, pcDNA3.1-LRPGC1, or pcDNA3.1- LRPGC1LKKAA/AAKYL (I). n = 12 (G), n = 11 (H), and n = 10 (I) per group. J, LA consumption after knockdown of Luciferase (control) or ERRγ by transfection with specific siRNAs in HepG2 cells (n = 10 per group). K, L, Kaplan-Meier survival analysis. Mice were injected ip with 1 g/kg body weight of LA following liver-targeted delivery of siRNA (30 μg/mouse) against Luciferase (n = 7) or Lrpgc1 (n = 6) through retro-orbital sinus (K), and mice were injected ip with 2 g/kg body weight of LA after ip preadministration of vehicle or DY131 (0.5 μmol/kg body weight) (n = 8 mice per group). Area under the curve (AUC) is shown on the Right. Values are shown as mean ± sem. Statistical analyses were performed by one- way ANOVA and Bonferroni/Dunn post hoc test (B, D, E, H, I), unpaired t test (G, J), or Logrank test (K, L). *P < .05, **P < .01

Article Snippet: To generate a PGC1 KO cell line, HepG2 cells were transfected with PGC1α CRISPR/Cas9 KO Plasmid (h) (sc-400070; Santa Cruz Biotechnology) and PGC1α HDR Plasmid (h) (sc400070-HDR; Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen).

Techniques: Transcription Assay, Transfection, Luciferase, Construct, Incubation, Expressing, Control, Activity Assay, Imaging, Microscopy, Fluorescence, Comparison, Knockdown, Injection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway.

doi: 10.1096/fj.202000492R

Figure Lengend Snippet: FIGURE 4 LRPGC1/ERRγ pathway activates mitochondrial function through induction of TFAM expression. A, Real time RT-PCR of HepG2 or PGC1 KO cells stimulated with lactic acid (LA) (final conc. 10 mM) for 1 hour (n = 4 for PDHA1 and PC, n = 3 for other genes). B, Living mitochondrial morphology. Mitochondria in HepG2 and PGC1 KO cells were labeled by Rhodamine 123 staining at 10 mM LA. Bar = 20 μm. C, Levels of genes downregulated in PGC1 KO cells in (A). Cells were transfected with pcDNA3.1-empty or pcDNA3.1-LRPGC1, and stimulated with LA (final conc. 10 mM) for 1.5 hours. Total RNA of the cells was then subjected to real time RT-PCR (n = 7 per group). D, TFAM expression level. PGC1 KO cells were transfected with pcDNA3.1-empty, pcDNA3.1-PGC1α, pcDNA3.1-LRPGC1, or pcDNA3.1- LRPGC1LKKAA/AAKYL and stimulated with LA (final conc. 10 mM) for 1.5 hours. Total RNA of the cells was then subjected to real time RT-PCR (n = 6 per group). E, Knockdown experiments. HepG2 cells were transfected with siRNA targeting Luciferase (control) or ERRγ, and stimulated with LA (final conc. 10 mM) for 1.5 hours. Total RNA was then subjected to real time RT-PCR (n = 9 per group). F, G, Western blotting with anti-TFAM antibody on whole cell lysates of PGC1 KO cells transfected with expression vectors including pcDNA3.1-empty, pcDNA3.1-PGC1α, pcDNA3.1- LRPGC1, or the LKKAA/AAKYL mutant of LRPGC1 (F), or transfected with siRNA targeting Luciferase or ERRγ (G). GAPDH was used as loading control. The experiments were repeated twice with similar results. H, Mitochondrial membrane potentials of PGC1 KO cells transfected with pcDNA3.1-empty, pcDNA3.1-PGC1α, or pcDNA3.1-LRPGC1, followed by incubation with 20 mM LA (n = 9 per group). I, Living mitochondrial morphology of PGC1 KO cells expressing CFP, CFP-PGC1α, or CFP-LRPGC1. After transfection, mitochondria were labeled by Rhodamine 123 staining at 10 mM LA. Bar = 20 μm. Values are shown as mean ± sem. Statistical analyses were performed by unpaired t test (A, C, E) or one-way ANOVA followed by Bonferroni/Dunn post hoc test (D, H). *P < .05, **P < .01. In panel (D), #P = .0764 versus PGC1α

Article Snippet: To generate a PGC1 KO cell line, HepG2 cells were transfected with PGC1α CRISPR/Cas9 KO Plasmid (h) (sc-400070; Santa Cruz Biotechnology) and PGC1α HDR Plasmid (h) (sc400070-HDR; Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Labeling, Staining, Transfection, Knockdown, Luciferase, Control, Western Blot, Mutagenesis, Membrane, Incubation

Journal: Cardiovascular Diabetology

Article Title: Mesenchymal stem cell transplantation for the infarcted heart: therapeutic potential for insulin resistance beyond the heart

doi: 10.1186/1475-2840-12-128

Figure Lengend Snippet: Regional insulin-stimulated cardiac glucose uptake. (a) Metabolic index of glucose uptake ( R g ) in the remote left ventricle and (b) peri-infarct region of the left ventricle. Cardiac R g values are relative to brain R g . n = 8-9 mice per group. (c) Remote left ventricle and (d) peri-infarct peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α), glucose transporter 4 (GLUT4) and hexokinase II (HKII) as determined by immunoblotting. (e) Left ventricle and (f) peri-infarct phospho-Akt (p-Akt), Akt and p-Akt-to-total Akt ratio (p-Akt/Akt) as determined by immunoblotting. (g) Representative immunoblotting performed to measure PGC-1α, GLUT4, HKII, p-Akt and Akt. Cardiac proteins are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content and are relative to the SHAM group. n = 6 mice per group. Data are mean ± S.E.M. *p < 0.05 vs. SHAM. †p < 0.05 vs. MI + PBS.

Article Snippet: Membranes were probed with peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α; Santa Cruz Biotechnology, Santa Cruz, CA, USA), glucose transporter 4 (GLUT4; Abcam, Cambridge, MA, USA), hexokinase II (HKII; Chemicon, Temecula, CA, USA), UCP3 (Abcam), phospho-Akt(Ser473) (p-Akt; Cell Signaling Technology, Whitby, ON, Canada) and Akt (Cell Signaling Technology) and oxidative phosphorylation complexes I-V (OXPHOS CI-CV; Abcam) antibodies.

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway

doi: 10.1096/fj.202000492r

Figure Lengend Snippet: FIGURE 1 LRPGC1 translocates into the nucleus following LA stimulation. A, Schematic structures of PGC1α and LRPGC1. AD, activation domain; RD, repression domain; NES, nuclear export signal; NLS, putative nuclear localization signal; Ub, ubiquitination. B, RT-PCR of Lrpgc1, Pgc1α, and Gapdh in indicated rat tissues. Data are from two rats. Upper, primer design. C, Western blotting on whole cell extracts of COS-1 cells expressing PGC1α or LRPGC1 incubated with MG132 (0-5 μM) for 6 hours. Anti-PGC1α antibody which recognizes N-terminus of PGC1 proteins was used. GAPDH was detected as loading control. Experiments were repeated three times with similar results. D, Confocal live images of CFP- PGC1α or CFP-LRPGC1 expressed in COS-1 cells. Bar = 20 μm. E, F, Time-lapse confocal imaging of COS-1 cells expressing CFP-LRPGC1. Notably, LRPGC1 translocated from the cytoplasm to the nucleus after treatment with lactic acid (LA) (final conc. 7.5 mM). Experiments were repeated more than 10 times with similar results. Bars = 50 (E) or 20 (F) μm. G, Western blotting on liver nuclear extract of the rats before and after injection of 2 g/kg body weight of LA (15, 30, 60 minutes). LRPGC1 was detected by anti-PGC1α antibody as described above. Anti-lamin B1 antibody was used as loading control. Experiments were repeated twice with similar results. Left, time-course schema

Article Snippet: To generate a PGC1 KO cell line, HepG2 cells were transfected with PGC1α CRISPR/Cas9 KO Plasmid (h) (sc-400070; Santa Cruz Biotechnology) and PGC1α HDR Plasmid (h) (sc400070-HDR; Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen).

Techniques: Activation Assay, Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Incubation, Control, Imaging, Injection

Journal: The FASEB Journal

Article Title: Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway

doi: 10.1096/fj.202000492r

Figure Lengend Snippet: FIGURE 3 LRPGC1 increases LA consumption through enhancement of ERRγ-mediated transcription. A, B, Transcription assay. COS-1 cells were transfected with pcDNA3.1-empty, pcDNA3.1-ERR (α, β, or γ), pcDNA3.1-LRPGC1, and ERRE-driven luciferase reporter construct (ERRE-Luc) and incubated with or without lactic acid (LA), as indicated. An actin promoter-driven β-galactosidase expression construct was transfected as an internal control. Luciferase activity was normalized by β-galactosidase activity (n = 4 per group). C, Time-lapse imaging of COS- 1 cells expressing CFP-LRPGC1 and YFP-ERRγ before and after treatment with LA (final conc. 7.5 mM). Bar = 20 μm. D-F, FRET microscopy on COS-1 cells expressing the indicated proteins. The cells were incubated with or without LA for 1 hour and fixed with 4% paraformaldehyde in 0.1M phosphate buffer for 10 minutes before FRET microscopy was performed. Bar = 10 μm. D, Ratio of YFP/CFP fluorescence intensity in ROIs under excitation at 458 nm (n = 34 for CFP-LRPGC1 + YFP-ERRγ, LA (−) and CFP-LRPGC1 + YFP, LA (+); n = 33 for CFP- LRPGC1 + YFP-ERRγ, LA (+)). E, Acceptor photobleaching. Increase of donor (CFP-LRPGC1, at 473 nm) fluorescence intensity was calculated after photobleaching of ROIs (YFP-ERRγ, at 514 nm) (n = 34 for CFP-LRPGC1 + YFP-ERRγ, LA (−) and CFP-LRPGC1 + YFP, LA (+); n = 33 for CFP-LRPGC1 + YFP-ERRγ, LA (+)). F, Pre- and post-bleached pseudocolor images. White circles denote the bleached ROI. Magnified ROI images are shown on the Right. G-I, Comparison of LA consumption between HepG2 and PGC1 KO cells (G), and among KO cells transfected with pcDNA3.1-empty, pcDNA3.1-PGC1α, or pcDNA3.1-LRPGC1 (H), or with pcDNA3.1-empty, pcDNA3.1-LRPGC1, or pcDNA3.1- LRPGC1LKKAA/AAKYL (I). n = 12 (G), n = 11 (H), and n = 10 (I) per group. J, LA consumption after knockdown of Luciferase (control) or ERRγ by transfection with specific siRNAs in HepG2 cells (n = 10 per group). K, L, Kaplan-Meier survival analysis. Mice were injected ip with 1 g/kg body weight of LA following liver-targeted delivery of siRNA (30 μg/mouse) against Luciferase (n = 7) or Lrpgc1 (n = 6) through retro-orbital sinus (K), and mice were injected ip with 2 g/kg body weight of LA after ip preadministration of vehicle or DY131 (0.5 μmol/kg body weight) (n = 8 mice per group). Area under the curve (AUC) is shown on the Right. Values are shown as mean ± sem. Statistical analyses were performed by one- way ANOVA and Bonferroni/Dunn post hoc test (B, D, E, H, I), unpaired t test (G, J), or Logrank test (K, L). *P < .05, **P < .01

Article Snippet: To generate a PGC1 KO cell line, HepG2 cells were transfected with PGC1α CRISPR/Cas9 KO Plasmid (h) (sc-400070; Santa Cruz Biotechnology) and PGC1α HDR Plasmid (h) (sc400070-HDR; Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen).

Techniques: Transcription Assay, Transfection, Luciferase, Construct, Incubation, Expressing, Control, Activity Assay, Imaging, Microscopy, Fluorescence, Comparison, Knockdown, Injection

Journal: The FASEB Journal

Article Title: Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway

doi: 10.1096/fj.202000492r

Figure Lengend Snippet: FIGURE 4 LRPGC1/ERRγ pathway activates mitochondrial function through induction of TFAM expression. A, Real time RT-PCR of HepG2 or PGC1 KO cells stimulated with lactic acid (LA) (final conc. 10 mM) for 1 hour (n = 4 for PDHA1 and PC, n = 3 for other genes). B, Living mitochondrial morphology. Mitochondria in HepG2 and PGC1 KO cells were labeled by Rhodamine 123 staining at 10 mM LA. Bar = 20 μm. C, Levels of genes downregulated in PGC1 KO cells in (A). Cells were transfected with pcDNA3.1-empty or pcDNA3.1-LRPGC1, and stimulated with LA (final conc. 10 mM) for 1.5 hours. Total RNA of the cells was then subjected to real time RT-PCR (n = 7 per group). D, TFAM expression level. PGC1 KO cells were transfected with pcDNA3.1-empty, pcDNA3.1-PGC1α, pcDNA3.1-LRPGC1, or pcDNA3.1- LRPGC1LKKAA/AAKYL and stimulated with LA (final conc. 10 mM) for 1.5 hours. Total RNA of the cells was then subjected to real time RT-PCR (n = 6 per group). E, Knockdown experiments. HepG2 cells were transfected with siRNA targeting Luciferase (control) or ERRγ, and stimulated with LA (final conc. 10 mM) for 1.5 hours. Total RNA was then subjected to real time RT-PCR (n = 9 per group). F, G, Western blotting with anti-TFAM antibody on whole cell lysates of PGC1 KO cells transfected with expression vectors including pcDNA3.1-empty, pcDNA3.1-PGC1α, pcDNA3.1- LRPGC1, or the LKKAA/AAKYL mutant of LRPGC1 (F), or transfected with siRNA targeting Luciferase or ERRγ (G). GAPDH was used as loading control. The experiments were repeated twice with similar results. H, Mitochondrial membrane potentials of PGC1 KO cells transfected with pcDNA3.1-empty, pcDNA3.1-PGC1α, or pcDNA3.1-LRPGC1, followed by incubation with 20 mM LA (n = 9 per group). I, Living mitochondrial morphology of PGC1 KO cells expressing CFP, CFP-PGC1α, or CFP-LRPGC1. After transfection, mitochondria were labeled by Rhodamine 123 staining at 10 mM LA. Bar = 20 μm. Values are shown as mean ± sem. Statistical analyses were performed by unpaired t test (A, C, E) or one-way ANOVA followed by Bonferroni/Dunn post hoc test (D, H). *P < .05, **P < .01. In panel (D), #P = .0764 versus PGC1α

Article Snippet: To generate a PGC1 KO cell line, HepG2 cells were transfected with PGC1α CRISPR/Cas9 KO Plasmid (h) (sc-400070; Santa Cruz Biotechnology) and PGC1α HDR Plasmid (h) (sc400070-HDR; Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Labeling, Staining, Transfection, Knockdown, Luciferase, Control, Western Blot, Mutagenesis, Membrane, Incubation

Journal: Pigment cell & melanoma research

Article Title: FBXW7 regulates a mitochondrial transcription program by modulating MITF

doi: 10.1111/pcmr.12704

Figure Lengend Snippet: (a) MITF protein levels were assayed using Western blotting (Pierce, Waltham, MA, USA) following transient transfection of either scrambled or FBXW7-specific siRNA in a panel of human melanoma cell lines. MM127, MM415, and MM485 harbor an NRASQ61 mutation whereas SH4, HT144, and A2058 melanoma lines have the BRAFV600E mutation. PGC-1alpha (Santa Cruz Biotechnology, Inc. Dallas, TX, USA) and PGC-1beta (Bethyl Laboratories, Inc. Montgomery, TX, USA) levels are shown. β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) was used as loading control. Densitometry is depicted in Figure S2.

Article Snippet: PGC-1alpha (Santa Cruz Biotechnology, Inc. Dallas, TX, USA) and PGC-1beta (Bethyl Laboratories, Inc. Montgomery, TX, USA) levels are shown. β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) was used as loading control.

Techniques: Western Blot, Transfection, Mutagenesis, Control