Journal: Neuron

Article Title: Nicotinamide pathway dependent Sirt1 activation restores calcium homeostasis to achieve neuroprotection in spinocerebellar ataxia type 7

doi: 10.1016/j.neuron.2019.11.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 80-90% confluent NPCs were transfected with PPARGC1A Human shRNA Plasmid Kit (TG310260, Origene) using Lipofectamine 3000 (ThermoFisher).

Techniques: Produced, Plasmid Preparation, Recombinant, Protease Inhibitor, Western Blot, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Calcium Assay, Staining, shRNA, Software

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: Generation of PGC1a stable KD cells The Colo357 pancreatic cancer cells with stable PGC1a KD and scrambled (SCR) control cells were generated using a PGC1a human shRNA plasmid kit (Origene, TG310260).

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: Generation of PGC1a stable KD cells The Colo357 pancreatic cancer cells with stable PGC1a KD and scrambled (SCR) control cells were generated using a PGC1a human shRNA plasmid kit (Origene, TG310260).

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: Generation of PGC1a stable KD cells The Colo357 pancreatic cancer cells with stable PGC1a KD and scrambled (SCR) control cells were generated using a PGC1a human shRNA plasmid kit (Origene, TG310260).

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: Generation of PGC1a stable KD cells The Colo357 pancreatic cancer cells with stable PGC1a KD and scrambled (SCR) control cells were generated using a PGC1a human shRNA plasmid kit (Origene, TG310260).

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: Generation of PGC1a stable KD cells The Colo357 pancreatic cancer cells with stable PGC1a KD and scrambled (SCR) control cells were generated using a PGC1a human shRNA plasmid kit (Origene, TG310260).

Techniques: Quantitative RT-PCR, Injection