Journal: Redox Biology

Article Title: Sirt1-Sirt3 axis regulates human blood-brain barrier permeability in response to ischemia

doi: 10.1016/j.redox.2017.09.016

Figure Lengend Snippet: Sirt1 regulates Sirt3 expression through AMPK-PGC1 pathway after OGD. Cells were transfected with siRNA for 48 h or pretreated with salermide (50 μM) for 30 min, and exposed to OGD. The expression of Sirt1 and Sirt3 was detected by western blot (A) and calculated (B and C). Cells were pretreated with salermide (50 μM) or compound C (5 μM) for 30 min and exposed to OGD. The expression of Sirt1 and Sirt3 was detected by western blot (D) and calculated (E and F). Cells were transfected with siRNA for 48 h with or without compound C, or pretreated with salermide (50 μM) for 30 min with or without compound C, and exposed to OGD. The expression of Sirt3 was detected by western blot (G). Cells were transfected with Si-control or Si-PGC1 for 48 h and exposed to OGD. The expression of Sirt3 was detected by western blot (H). Data are shown as mean ± SEM. * p < 0.05 vs. Control. # p < 0.05 vs. OGD. & p < 0.05 vs. Si-control. $ p < 0.05.

Article Snippet: The specific siRNA targeted Sirt1 (Si-Sirt1, sc-40986), Sirt3 (Si-Sirt3, sc-61555), PGC1 (Si-PGC1, sc-38884) and control siRNA (Si-Control, sc-37007), which should not knock down any known proteins, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Cell Death & Disease

Article Title: PGC-1α protects against MASH via Tim23-dependent inhibition of DRP1-mediated ferroptosis

doi: 10.1038/s41419-026-08493-8

Figure Lengend Snippet: Primary hepatocytes were transfected with negative control (NC) or PGC-1α CRISPR activation plasmid (PGC-1α CRISPR ACT) and treated with PA, leading to four groups of the Act-NC, Act-NC + PA, Act-PGC-1α and Act-PGC-1α + PA. A Oil Red O, Perls’ Blue staining and Tim23 Immunohistochemistry. B , B ’ Western blot analyses of the relative levels of hepatic PGC-1α, Tim23, Drp1, P-Drp1 Ser616 , ACSL4, GPX4 and FTH1 to GAPDH. C RT-qPCR analyses of the relative levels of PGC-1α, Drp1, ACSL4, GPX4, TFR1 and FTH1 mRNA transcripts. D The GSH levels. E Images of MitoTracker and MitoSOX staining. F Images of C11-BODIPY 581/591 staining. Data are representative images or expressed as the mean ± SD of each group ( n = 3) from at least three independent treatments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Special reagents included primary antibodies against Drp1, Nrf1, P-Drp1ser616, alpha-smooth muscle actin (α-SMA), collagen I, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c oxidase subunit 4 (COXIV) and cleaved caspase-3 (Cell Signaling Technology, Beverly, USA); PGC-1α, ACSL4, tumor necrosis factor (TNF)α, Hepatocyte nuclear factor 4-alpha (HNF-4α), Desmin and interleukin (IL)-6 (Abcam, Cambridge, USA); Tim23 (Santa, TX, USA); glutathione peroxidase 4 (GPX4), Ferroton heavy chain 1 (FTH1), transferrin receptor 1 (TFR1) and F4/80 (Proteintech, Wuhan, China); Lymphatic Vessel Endothelial Receptor-1(Lyve-1) (ABclonal, Wuhan, China); Nrf2, P-MLKL, GSDMD-N (HUABIO, Hangzhou, China); special kits for hematoxylin and eosin (H&E), Sirius Red, and Oil Red staining (Solarbio, Beijing, China); immunohistochemical staining kit (Maixin Biological Technology, Fujian, China); the kits for measurements of alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), glucose, glutathione (GSH), malondialdehyde (MDA), and iron contents (Jiancheng Biological Engineering Institute, Nanjing, China); enzyme-linked immunosorbent assay (ELISA) kits for measurements of insulin (Crystal Chem, Chicago, USA); Tissue mitochondria isolation kit (Beyotime, Shanghai, China); Perls’ blue (Sbjbio, Nanjing, China); PGC-1α clustered regularly interspaced short palindromic repeats (CRISPR) activation plasmid (sc-400070-ACT) and Protein A/G plus-agarose (Santa Cruz Biotechnology, CA, USA); PGC-1α and Drp1 small interfering RNAs (siRNAs) (LIKELI, Beijing, China); dual luciferase assay kits and TnT® quick coupled transcription/ translation systems (Promega, Wisconsin, USA); Lipofectamine 2000, C-11 BODIPY 581/591, MitoSOX and Mitotracker (Invitrogen, CA, USA); JC-1 (Chemodex, SG, SUI); Ferrostatin-1 (Fer-1) (MedChemExpress, NJ, USA).

Techniques: Transfection, Negative Control, CRISPR, Activation Assay, Plasmid Preparation, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR

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: Clinical Cancer Research

Article Title: PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions

doi: 10.1158/1078-0432.ccr-20-5020

Figure Lengend Snippet: Figure 1. Meta-analysis of stemness programs and metabolic states in IPMN- and PanIN-mediated PDAC progression. NCBI GEO datasets (GSE19650 and GSE43288) were used to investigate the differential transcriptomic signatures of stemness and metabolic genes. The data analysis and processing were performed by quantile normalization and log2 transformation. A–N, Representation of the differentially expressed glycolysis genes (A–G), OXPhos genes (H–K), MYC (L), PPARGC1A (M), and CPT2 (N) in indicated samples: NP (N ¼ 7), IPMN-derived PDAC (IPMN-PDAC; n ¼ 3), IPMA or IPMN with low-grade dysplasia (n ¼ 6), and IPMC or IPMN with high- grade dysplasia (n ¼ 6). O–X, Representation of the differentially expressed glycolysis genes (O–U), fatty acid b-oxidation genes (V–W), and PPARGC1A (X) in indicated samples: NP (n ¼ 3), PanIN (n ¼ 13), and PDAC (n ¼ 4). Data represent mean SD. P values were calculated using ordinary one-way ANOVA (multiple comparisons). The mean of each sample was compared with the mean of NP. Asterisks indicate a statistically significant difference between each sample and NP (P < 0.05; , P < 0.05; , P < 0.01; P < 0.001.) Y, Venn diagram showing common and unique overexpressed stemness genes in PanIN and IPMN. Data represent mean SD. P valueswere calculated using ordinary one-way ANOVA (multiple comparisons; ,P < 0.05). Z, Network analysis of the differentially expressed stemness and metabolic genes from the GSE19650 dataset using IPA. The network shows that the PPARGC1A is central to stemness, FAO, and OXPhos pathways in IPMN.

Article Snippet: LGKC1 cells with stable KD of PGC1a were generated using a mouse PGC1a shRNA plasmid (Santa Cruz Biotechnology, catalog no. Sc-38885-SH) and control shRNA plasmid B (Sc-108065).

Techniques: Transformation Assay, Derivative Assay

Journal: Clinical Cancer Research

Article Title: PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions

doi: 10.1158/1078-0432.ccr-20-5020

Figure Lengend Snippet: Figure 2. Differential expression of metabolic regulators, PGC1a and CPT1A, in different stages of PDAC development. A–F, IHC analysis of PGC1a (A–C) and CPT1A (D–F) in indicated samples. A histoscore was calculated by multiplying intensity and positivity. Data represent mean SD. P values were calculated using ordinary one-way ANOVA (multiple comparisons). The mean of each sample was compared with the mean of NP. Asterisks indicate a statistically significant difference between each sample and NP (, P < 0.05; , P < 0.01; , P < 0.001). Scale bar 200 mm. C and F, Magnified PanIN2 and IPMN regions duplicated from the original PanIN2 and IPMN IHC images of A and D to show the subcellular localization of PGC1a (C) and CPT1A (F) were shown.

Article Snippet: LGKC1 cells with stable KD of PGC1a were generated using a mouse PGC1a shRNA plasmid (Santa Cruz Biotechnology, catalog no. Sc-38885-SH) and control shRNA plasmid B (Sc-108065).

Techniques: Quantitative Proteomics

Journal: Clinical Cancer Research

Article Title: PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions

doi: 10.1158/1078-0432.ccr-20-5020

Figure Lengend Snippet: Figure 4. ADM/PanIN and IPMN show upregulation of PGC1a and display unique metabolic states. A and B, IHC analysis of PGC1a in PBS- or cerulean-treated KC pancreas samples. The histogram to the right shows the histoscore of PGC1a. Data represent mean SD (n ¼ 3). Scale bar 200 mm. C and D, qRT- PCR analysis of PPARGC1A and CPT1A in indicated samples. The PCR data were normalized with the Actb gene. Data represent mean SD (n ¼ 3). E and F, Maximal respiration and spare respiratory capacity reflected by OCR were measured using the Seahorse extracellular flux analyzer. Data are mean SEM (n ¼ 6). G and H, Glycolysis and glycolytic capacity reflected by ECAR was measured in indicated samples using Seahorse extracellular flux analyzer. Data are mean SEM (n ¼ 6). I, Maximal endogenous OCR due to FAO measured by XF Palmitate-BSA FAO Substrate with the XF Cell Mito Stress Test kit using the Seahorse extracellular flux analyzer. Data are mean SEM (n ¼ 3). J and K, Immunofluorescence images of pancreas harvested from PBS- or cerulean-treated KC mice stained with PNA-Rhodamine, DBA-FITC, UEA1-FITC, CD133, PGC1a, CPT1A, and DAPI (as indicated). Scale bar 100 mm. L and M, qRT- PCR analysis of indicated genes in indicated samples. The PCR data were normalized with the Actb gene. Data represent mean SD (n ¼ 3). N, Basal OCR was measured in acinar, AD, and ductal populations using XF Cell Mito Stress Test kit using the Seahorse extracellular flux analyzer. Data are mean SEM (n ¼ 3). O, Immunofluorescence images of pancreas harvested from 10-week-old KC and WT mice stained with CD133, PGC1a, cKIT, and DAPI (as indicated). Scale bar 50 mm. P, qRT- PCR analysis of indicated genes in LGKC1 control and doxycycline (Dox)-induced samples. The PCR data were normalized with the Actb gene. Data represent mean SD (n ¼ 3). Q, OCR was measured following the addition of oligomycin (O; 1 mmol/L), FCCP (F; 0.5 mmol/L), and electron transport inhibitor rotenone/antimycin A (R/A; 0.5 mmol/L). Data are mean SD (n ¼ 6). R, ECAR was measured following the addition of glucose (Glc; 10 mmol/L), oligomycin (O; 1 mmol/L), and 2-deoxyglucose (2DG; 50 mmol/L). Data are mean SEM (n ¼ 6). S, Immunofluorescence images of human IPMN organoids stained with PGC1a, CPT1A, and DAPI (as indicated). Scale bar 50 mm.

Article Snippet: LGKC1 cells with stable KD of PGC1a were generated using a mouse PGC1a shRNA plasmid (Santa Cruz Biotechnology, catalog no. Sc-38885-SH) and control shRNA plasmid B (Sc-108065).

Techniques: Quantitative RT-PCR, Staining, Control

Journal: Clinical Cancer Research

Article Title: PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions

doi: 10.1158/1078-0432.ccr-20-5020

Figure Lengend Snippet: Figure 5. ADM/PanIN and IPMN show the upregulation of unique PGC1a-interacting partners. A, Protein–protein interactions analysis of PPARGC1A using “STRING” software. B–I, A meta-analysis of genes that encode PGC1a-interacting proteins using the human IPMN progression dataset GSE19650. Datasets were processed using standard GEO2R analysis, followed by quantile normalization and log2 transformation. Data represent mean SD. P values were calculated using ordinary one-way ANOVA (multiple comparisons). The mean of each sample was compared with the mean of NP. Asterisks indicate a statistically significant difference between each sample and NP. J, qRT-PCR analysis of indicated genes in acinar and AD cells. The PCR data were normalized with the Actb gene. Data represent mean SD (n ¼ 3). K and L, Immunofluorescence images with PGC1a, PPARg, and DAPI staining on indicated samples. M and N, Immunofluorescence images. NRF1 staining along with DAPI on pancreatic tissues harvested from control (KC) and cerulean-treated KC (KCþCer) mouse (M). The KCþCer immunofluorescence image, which was shown for NRF1 staining in M (bottom image), was further showed for the co-expression of NRF1 with PGC1a (N, bottom images). The co-expression of NRF1 with PGC1a was shown in another KCþCer tissue section (N, top images). O, Immunofluorescence images with PGC1a, NRF1, and DAPI staining on indicated samples. Scale bar 50 mm. For all histograms, P values were calculated by Student t test (, P < 0.05; , P < 0.01; , P < 0.001.)

Article Snippet: LGKC1 cells with stable KD of PGC1a were generated using a mouse PGC1a shRNA plasmid (Santa Cruz Biotechnology, catalog no. Sc-38885-SH) and control shRNA plasmid B (Sc-108065).

Techniques: Protein-Protein interactions, Software, Transformation Assay, Quantitative RT-PCR, Staining, Control, Expressing

Journal: Clinical Cancer Research

Article Title: PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions

doi: 10.1158/1078-0432.ccr-20-5020

Figure Lengend Snippet: Figure 6. PGC1a-mediated OXPhos and FAO-OXPhos regulate stemness in ADM/PanIN and IPMN, respectively. A, qRT-PCR analysis of Ppargc1a in the scramble and PGC1a KD in LGKC1 cells. The PCR data were normalized with the Actb gene. Data represent mean SD (n ¼ 3). B, LGKC1 SCR and PGC1a KD cells were injected subcutaneously into nude mice and maintained with doxycycline (DOX) in water. The subcutaneous tumors were excised 21 days after implantation, followed by the measurement of tumor volume and weight (bar graphs). Data are mean SD (n ¼ 4). The significance was determined by a t test (, P < 0.05; , P < 0.01; , P < 0.001). C, qRT-PCR analysis of indicated genes in the scramble and PGC1a KD LGKC1þDOX cells. The PCR data were normalized with the Actb gene. Data represent mean SD (n ¼ 3). D–F, Basal, maximal respiration, and spare respiratory capacity reflected by OCR due to FAO measured by XF Palmitate-BSA FAO Substrate with the XF Cell Mito Stress Test kit using the Seahorse extracellular flux analyzer. Data are mean SEM (n ¼ 3). G and H, Morphology of human IPMN organoids growing in the presence and absence of SR18292. (Continued on the following page.)

Article Snippet: LGKC1 cells with stable KD of PGC1a were generated using a mouse PGC1a shRNA plasmid (Santa Cruz Biotechnology, catalog no. Sc-38885-SH) and control shRNA plasmid B (Sc-108065).

Techniques: Quantitative RT-PCR, Injection

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