Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) Western blots showing inducible expression of MPC1 and MPC2 at 2-hr intervals following treatment with 1 μg/ml doxycycline. Citrate synthase and tubulin were used as loading controls. Both endogenous and epitope tag bands are shown. ( b ) Quantification of the 2D area of HepG2 cells with MPC expression (MPC+) or empty vector (EV) fixed and stained with rhodamine phalloidin at the indicated times after doxycycline treatment. Data are presented as mean ± s.d. from five biological replicates, with each replicate representing the average size of 25 randomly selected cells. ( c ) Quantification of the 2D area of MPC+ or EV HepG2 cells treated with 10 μM UK5099. Data are presented as mean ± s.d. from five biological replicates, with each replicate representing the average size of 25 randomly selected cells. ( d ) Representative brightfield images of HepG2 spheroids with empty vector (EV) or MPC expression (MPC+) treated with 1 μg/ml doxycycline for 6 days. The scale bar represents 200 μm. ( e ) Quantification of spheroid area from images of MPC+ or EV HepG2 spheroids, with or without doxycycline treatment. Data are presented as mean ± s.d. from 30 technical replicates. ( f ) Forward scatter (FSC) of cells dissociated from MPC+ (red) or EV spheroids with cell count normalized to mode. ( g ) Median FSC of MPC+ HepG2 spheroids treated with or without 1 μg/ml doxycycline. Data are presented as mean ± s.d. from three biological replicates. ( h ) Fold change in macromolecules—DNA, TAGs, RNA, and protein—fractionated from EV or MPC+ HepG2 spheroids normalized to that in EV HepG2 cells. ( i ) Representative images showing L-homopropargylglycine (HPG)-labeled newly synthesized proteins in EV or MPC+ HepG2 cells. The top panels show HPG staining, and the lower panels show nuclei stained with DAPI. The scale bar represents 20 μm. ( j ) Quantification of HPG fluorescence intensity is presented as mean ± s.d. from 35 cells, for both EV and MPC+ cells. ( k ) Western blot analysis of nascent protein synthesis using a puromycin incorporation assay (20 μg/ml puromycin for 30 min) in either EV or MPC+ HepG2 spheroids (protein lysates from 16 spheroids loaded in each lane). ( l ) Quantification of 70 kD band intensity in puromycin blot normalized with tubulin band intensity and represented as mean ± s.d. from three independent experiments. ( m ) Relative accumulation of destabilized GFP (d2GFP) in EV or MPC+ HepG2 spheroids treated with or without 1 μg/ml doxycycline ± 10 μM UK5099. Data are presented as mean ± s.d. from three biological replicates. Unpaired t -tests, one-way, or two-way ANOVA tests were performed to evaluate the statistical significance of the data, with p-values noted in the graph if significance was observed. Figure 3—source data 1. Source data related to . Figure 3—source data 2. Original files for western blot analysis displayed in . Figure 3—source data 3. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 4. Original files for western blot analysis displayed in . Figure 3—source data 5. PDF files containing original western blots for , indicating the relevant bands and treatments.

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Western Blot, Expressing, Plasmid Preparation, Staining, Cell Counting, Labeling, Synthesized, Fluorescence

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) Quantification of cell volume from confocal images of HepG2 cells expressing MPC (MPC+) or an empty vector (EV). Data is presented as mean ± s.d. from 22 EV cells and 25 MPC+ cells. ( b ) Quantification of the number of cells per spheroid in EV or MPC+ HepG2 cells with or without doxycycline treatment. Data is presented as mean ± s.d. from 18 technical replicates. ( c ) Cell cycle profiles of MPC+ HepG2 spheroids treated with or without doxycycline treatment. Data is presented as mean ± s.d. from three biological replicates. ( d ) Annexin/Propidium Iodide (PI) analysis of MPC+ HepG2 cells cultured with or without doxycycline. Data is presented as mean ± s.d. from three biological replicates. Concentrations of ( e ) DNA, ( f ) RNA, ( g ) triacylglycerides (TAGs) and ( h ) protein measured for 18 MPC+ and 18 EV HepG2 spheroids. Data is shown as mean ± s.d. from three biological replicates. Unpaired t -tests and one-way ANOVA tests were performed to evaluate the statistical significance of the data, with p-values mentioned in the graph if significance is noted. Figure 3—figure supplement 1—source data 1. Source data related to .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Expressing, Plasmid Preparation, Cell Culture

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) Schematic illustration of the 13 C-glucose tracing strategy used to measure the activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). TCA metabolites labeled with two heavy carbons ( 13 C or M+2 TCA pool) result from PDH activity, whereas M+3 TCA metabolites result from PC activity. PDH is inhibited by PDK-mediated phosphorylation. DCA and AZD7545 are inhibitors of PDK. ( b ) Fractional enrichment of M+2 succinate in empty vector (EV; red) and MPC-expressing (MPC+; blue) HepG2 cells at the indicated times after 13 C-glucose tracing. MPC expression was induced for 24 hr by treatment with 1 µg/ml doxycycline and media was changed to 12 C-glucose. The change in M+2 succinate is significant (by two-way ANOVA test) at 1 hr after 13 C glucose incubation. ( c ) Fractional enrichment of M+3 succinate in EV (red) and MPC+ (blue) HepG2 at the indicated times after 13 C-glucose tracing. MPC expression was induced for 24 hr by treatment with 1 μg/ml doxycycline. The change in M+3 succinate is significant (by two-way ANOVA test) at 4 hr after 13 C-glucose incubation. ( d ) Rate of oxygen consumption (OCR) in EV and MPC+ HepG2 cells. ( e ) Representative images of phalloidin- and DAPI-stained fat body cells. Arrows indicate GFP-positive clones with MPC expression (MPC+), Pcb knockdown with MPC expression (MPC+, Pcb -KD), or Pcb overexpression ( Pcb +). The scale bar represents 20 µm. ( f ) Quantification of the area of GFP-positive clones with control, MPC+, Pcb over-expression ( Pcb +), Pcb and MPC co-expression (MPC+, Pcb +), Pcb knockdown ( Pcb -KD), and Pcb knockdown with MPC expression (MPC+, Pcb -KD) shown as mean ± s.d. of five biological replicates, with each group representing the analysis of 20 the indicated clonal cells. Concentration of glucose in the fat body ( g ) and concentration of glucose and trehalose in hemolymph ( h ) of larva with fat body-specific expression MPC or control. Data is presented as mean ± s.d. of three biological replicates analyzed by unpaired t -tests. ( i ) Schematic illustration of the strategy to analyze gluconeogenesis from 13 C-lactate. Cells convert 13 C-lactate into 13 C-pyruvate, which is transported into mitochondria by the MPC. PC converts 13 C-pyruvate (M+3) into oxaloacetate (M+3). PEPCK2 converts oxaloacetate (M+3) into phosphoenolpyruvate (M+3), which is converted into M+6 glucose and excreted from cells. ( j ) Relative abundances of M+3 phosphoenolpyruvate (PEP) in EV and MPC+ HepG2 cells and ( k ) M+6 glucose in their respective media following treatment with 20 mM 13 C-lactate for 4 hr. Data is presented as mean ± s.d. of three biological replicates, each with an average of three technical replicates. ( l ) Representative brightfield images of EV, MPC+, and PC knockout (KO) or PEPCK2 KO with or without MPC expression HepG2 spheroids. The scale bar represents 200 µm. ( m ) Quantification of spheroid area is presented as mean ± s.d. of 30 technical replicates. ( n ) Representative images of phalloidin- and DAPI-stained fat body cells. Arrows indicate GFP-positive clones with MPC expression (MPC+), and Pepck2 knockdown with MPC expression (MPC+, Pepck2 -KD). The scale bar represents 20 µm. ( o ) Quantification of the area of GFP-positive clones with MPC+, Pepck2 knockdown ( Pepck2 -KD), Pepck2 knockdown with MPC+ (MPC+, Pepck2 -KD), Fbp knockdown ( Fbp -KD), Fbp knockdown with MPC+ (MPC+, Fbp -KD). Data is presented as mean ± s.d. of five biological replicates, with each group analyzing 20 clonal cells of the mentioned genetic manipulations. ( p ) Representative images of fat body clones stained with OPP (red). Arrows indicate GFP-positive clones with MPC expression (MPC+), Pcb knockdown with MPC expression (MPC+, Pcb -KD), or Pepck2 knockdown with MPC expression (MPC+, Pepck2 -KD). The scale bar represents 20 µm. ( q ) Quantification of OPP intensity in the indicated clones compared with adjacent wild-type cells. Data is presented as mean ± s.d. Unpaired t -tests, one-way ANOVA tests, or two-way ANOVA tests were performed to evaluate the statistical significance of the data, with p-values mentioned in the graph if significance is noted. Panel a was created with BioRender.com . Panel i was created with BioRender.com . Figure 4—source data 1. Source data related to .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Activity Assay, Labeling, Phospho-proteomics, Plasmid Preparation, Expressing, Incubation, Staining, Clone Assay, Knockdown, Over Expression, Control, Concentration Assay, Knock-Out

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: Fractional enrichment and metabolite counts of M+3 3-phosphoglyceric acid ( a ), M+3 pyruvate ( b ), and M+3 alanine ( c ) in empty vector (EV; red) and MPC expressing (MPC+; blue) HepG2 cells at the indicated times after 13 C glucose tracing. Metabolite counts of M+2 succinate ( d ) and M+3 succinate ( e ) in EV (red) and MPC+ (blue) HepG2 cells. Fractional enrichment and metabolite counts of M+2 fumarate ( f ), M+2 malate ( g ) M+3 fumarate ( h ), and M+3 malate ( i ) in EV (red) and MPC+ (blue) HepG2 cells. A two-way ANOVA test showed significant differences at 4 hr after 13 C glucose tracing. Figure 4—figure supplement 1—source data 1. Source data related to .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Plasmid Preparation, Expressing

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) NADH/NAD + ratios from the cytoplasmic and mitochondrial fractions of control and MPC-expressing (MPC+) fat bodies. Data is presented as mean ± s.d. of six biological replicates. ( b ) NADH/NAD + ratios from the cytoplasmic and mitochondrial fractions of empty vector (EV) and MPC-expressing (MPC+) HepG2 spheroids. Data is presented as mean ± s.d. of six biological replicates. ( c ) Representative images of phalloidin- and DAPI-stained fat bodies. Arrows indicate GFP-positive cells with either MPC+ or MPC and Nmnat co-expression (MPC+, Nmnat +). The scale bar represents 20 µm. ( d ) Quantification of the areas of GFP-positive cells with control, MPC+, Nmnat overexpression ( Nmnat +), MPC and Nmnat co-expression (MPC+, Nmnat +). Data is presented as mean ± s.d. of five biological replicates, with 20 clonal cells analyzed for each of the indicated genetic manipulations. ( e ) Representative images of Phalloidin and DAPI-stained fat body tissues. Arrows indicate GFP-positive cells showing MPC expression (MPC+); NDI and MPC expression (MPC+, NDI+); and NDX and MPC expression (MPC+, NDX+). The scale bar represents 20 µm. ( f ) Quantification of the area of GFP-positive cells with control, MPC+, NDI expression (NDI+), NDI and MPC expression (MPC+, NDI+), NDX expression (NDX+), NDX and MPC co-expression (MPC+, NDX+). Data is presented as mean ± s.d. of five biological replicates, with 20 clonal cells analyzed for each of the indicated genetic manipulations. ( g ) Representative bright field images of EV or MPC+HepG2 spheroids cultured with NAD + supplements (100 nM nicotinamide riboside or 1 µM NMN) as indicated. The scale bar represents 200 µm. ( h ) Quantification of spheroid area is presented as mean ± s.d. of 30 technical replicates. ( i ) Representative images MPC+ or MPC+, NDI+ GFP-positive clones and of fat body cells stained with OPP (bottom). Clonal cells are mapped with dotted lines. The scale bar represents 20 µm. ( j ) Fold change in OPP intensity of 35 GFP-positive cells compared with adjacent wild-type cells. Data is presented as mean ± s.d. ( k ) Relative accumulation of destabilized GFP (d2GFP) in spheroids of EV or MPC+ HepG2 cells treated with NAD + supplements (100 nM nicotinamide riboside or 1 µM NMN) as indicated. Unpaired t-tests, one-way ANOVA tests, or two-way ANOVA tests were performed to assess the statistical significance of the data, with p-values indicated in the graph where significance was observed. Figure 5—source data 1. Source data related to .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Control, Expressing, Plasmid Preparation, Staining, Over Expression, Cell Culture, Clone Assay

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: qRT-PCR showing ( a ) Mpc1 , ( b ) Mpc2 , and ( c ) pcb transcripts from fat bodies of control, MPC+, Pcb over-expression ( Pcb +), Pcb and MPC co-expression (MPC+, Pcb +), Pcb knock down ( Pcb -KD) and Pcb knock down with MPC expression (MPC+, Pcb -KD) normalized to rp49 transcript abundance. ( d ) Quantification of the area of GFP-positive fat body clones with control, MPC expression (MPC+), Pcb knockout ( Pcb KO ), or MPC expression with Pcb knockout (MPC+, Pcb KO). Data is presented as mean ± s.d. of five biological replicates, with each group analyzing 20 clonal cells of the indicated genetic manipulations. ( e ) Quantification of the areas of HepG2 cells with MPC+ with or without pyruvate carboxylase (PC) knockout using two independent sgRNA guides (PCg5 and PCg6). Western blots show the efficiency of PC knockout. Data is presented as mean ± s.d. of five biological replicates, each representing 25 randomly selected cells for each indicated genotype. qRT-PCR showing ( f ) Mpc1 , ( g ) Mpc2 , ( h ) Pdha , and ( i ) Dlat transcripts from fat bodies of control, MPC+, Pdha knockdown ( Pdha -KD), Pdha knockdown with MPC expression (MPC+, Pdha- KD), Dlat knockdown ( Dlat -KD), Dlat knockdown with MPC expression (MPC+, Dlat -KD) normalized to rp49 transcript abundance. ( j ) Representative images of phalloidin- and DAPI-stained fat body cells. Arrows indicate GFP-positive clones with MPC expression (MPC+), Pdha knockdown ( Pdha -KD), or MPC expression with Pdk knockdown (MPC+, Pdk -KD). The scale bar represents 20 µm. Right. ( k ) Quantification of the areas of GFP-positive clones with control, MPC+, Pdha knockdown ( Pdha -KD), Pdha knockdown with MPC expression (MPC+, Pdha- KD), Dlat knockdown ( Dlat -KD), Dlat knockdown with MPC expression (MPC+, Dlat -KD); Pdk knockdown ( Pdk -KD), and Pdk knockdown with MPC expression (MPC+, Pdk -KD). Data is presented as mean ± s.d. of five biological replicates, with 20 clonal cells analyzed for each of the indicated genetic manipulations. ( l ) Quantification of the areas of MPC+ HepG2 cells treated with the PDK inhibitors AZD7545 (10 µM) or dichloroacetate (1 mM). Data is presented as mean ± s.d. of five biological replicates, with 25 randomly selected cells analyzed for each of the indicated groups. ( m ) Quantification of the areas of MPC+, PEPCK2 knockout HepG2 cells. Western blots show the efficiency of PEPCK2 knockout. Data is presented as mean ± s.d. of five biological replicates with 25 randomly selected cells analyzed for each of the indicated groups. qRT-PCR showing ( n ) Mpc1 , ( o ) Mpc2 , ( p ) Pepck2 , and ( q ) Fbp transcripts from fat bodies of MPC+, Pepck2 knockdown ( Pepck2 -KD), Pepck2 knockdown with MPC+ (MPC+, Pepck2 -KD), Fbp knockdown ( Fbp -KD), Fbp knockdown with MPC+ (MPC+, Fbp -KD) normalized to rp49 transcript abundance. ( r ) Quantification of the area of GFP-positive control; MPC+; Pepck2 KO ; and MPC+, Pepck2 KO fat body clonal cells. Data is presented as mean ± s.d. of five biological replicates, with 20 clonal cells analyzed for each of the indicated genetic manipulations. Unpaired t -tests, one-way ANOVA tests, or two-way ANOVA tests were performed to evaluate the statistical significance of the data, and p-values are mentioned in the graph if significance is noted. Figure 4—figure supplement 2—source data 1. Source data related to . Figure 4—figure supplement 2—source data 2. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 3. Original files for western blot analysis displayed in . Figure 4—figure supplement 2—source data 4. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 5. Original files for western blot analysis displayed in .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Quantitative RT-PCR, Control, Over Expression, Expressing, Knockdown, Clone Assay, Knock-Out, Western Blot, Staining, Positive Control

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) A schematic illustration of mitochondrial pyruvate metabolism and gluconeogenesis. Following are the abbreviations: PEP: phosphoenolpyruvate, F1,6BP: fructose-1, 6-bisphosphate, F6P: fructose-6-phosphate, G6P: glucose-6-phosphate, F6Pase: fructose bisphosphatase, and G6PC: glucose-6-phosphatase ( b ) Western blot analysis shows the expression levels of phosphorylated PDH (p-PDH), total PDH, G6PC, PEPCK2, PC, and tubulin in MPC-expressing (MPC+) HepG2 cells treated with 1 µg/ml doxycycline at the indicated time points. ( c ) Schematic illustrating the regulation of PDH, PC, and gluconeogenesis by a network of cofactors and substrates. Following are the abbreviations: 1,3 BPG: 1,3-bisphosphoglycerate, G3P: glucose-3-phosphate, PGK: phosphoglycerate kinase, and GAPDH: glyceraldehyde-3-phosphate dehydrogenase. ( d ) Fold enrichment of acetyl-CoA in EV and MPC+ HepG2 cells. Data is presented as mean ± s.d. of three biological replicates using unpaired t -tests. ( e ) The ratio of ATP to ADP in EV and MPC+ HepG2 cells. Data is presented as mean ± s.d. of three biological replicates using unpaired t -tests. Cellular concentrations of NADH ( f ) and NAD + ( g ) and NADH/NAD + ratios ( h ) in EV and MPC+ HepG2 cells 24 hr after treatment with 1 µg/ml doxycycline. Data is presented as mean ± s.d. of three biological replicates using unpaired t -tests. ( i ) Quantification of the areas of MPC+ HepG2 cells cultured with 2 nM gramicidin. Data is presented as mean ± s.d. of five biological replicates, with 25 randomly selected cells analyzed for each of the indicated groups. ( j ) Representative bright field images of EV or MPC+ HepG2 spheroids with NDI expression (NDI+). The scale bar represents 200 µm. ( k ) Quantification of spheroid areas is presented as mean ± s.d. of 30 technical replicates. Western blots show the efficiency of MPC expression. ( l ) Quantification of the area of MPC-expressing HepG2 cells cultured with or without 100 nM duroquinone. Data is presented as average ± s.d. of five biological replicates, with each group consisting of 25 randomly selected cells. Data is presented as mean ± s.d. of three biological replicates. Unpaired t-tests, one-way ANOVA, or two-way ANOVA tests were performed to assess the statistical significance, with p-values indicated in the graph where significance was noted. Panel a was created with BioRender.com . Panel c was created with BioRender.com . Figure 5—figure supplement 1—source data 1. Source data related to . Figure 5—figure supplement 1—source data 2. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 5—figure supplement 1—source data 3. Original files for western blot analysis displayed in . Figure 5—figure supplement 1—source data 4. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 5—figure supplement 1—source data 5. Original files for western blot analysis displayed in .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Western Blot, Expressing, Cell Culture

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) A heatmap of the abundances of amino acids in empty vector (EV) and MPC expressing (MPC+) HepG2 cells cultured under standard conditions. Color codes indicate relative abundances for each amino acid: blue (low), green (similar), and yellow (high). ( b ) Representative bright field images of EV or MPC+ HepG2 spheroids cultured with 2x or 3x the recommended concentration of non-essential amino acids cocktail (NEAA). The scale bar represents 200 µm. ( c ) Quantification of spheroid areas from EV and MPC+ HepG2 spheroids cultured with 2x or 3x NEAA. Data is presented as mean ± s.d. of 30 technical replicates. ( d ) Representative images of phalloidin- and DAPI-stained fat body cells from animals fed a standard diet or a diet supplemented with 5x NEAA. Arrows indicate GFP-positive clones with MPC expression (MPC+). The scale bar represents 20 µm. ( e ) Quantification of the area of MPC+ fat body clonal cells. Data is presented as mean ± s.d. of five biological replicates, with each data point representing the average size of 20 clones collected from five male larvae. ( f ) Representative images of phalloidin- and DAPI-stained fat body cells. Arrows indicate GFP-positive MPC+ clones and MPC+ and slimfast over-expression (MPC+, Slif +). The scale bar represents 20 µm. ( g ) Quantification of the area of GFP-positive clones with control, MPC expression, Slimfast overexpression ( Slif +) and MPC+ clones with Slimfast overexpression (MPC+, Slif +). Data presented as mean ± s.d. of five biological replicates, with each 20 clonal cells analyzed for each of the indicated genetic manipulations. ( h ) Quantification of the cell areas of EV or MPC+ HepG2 cells cultured under standard conditions or with excess of the indicated amino acid—10 mM glycine, 5 mM alanine, 5 mM serine, 5 mM asparagine, 5 mM aspartic acid, 5 mM glutamic acid, or 5 mM proline. Data is presented as mean ± s.d. of five biological replicates. ( i ) Representative images of phalloidin- and DAPI-stained fat body cells. Arrows indicated GFP-positive cells with MPC expression (MPC+), Got2 knockdown ( Got2 KD), and Got2 overexpression with MPC expression (MPC+, Got2 +). ( j ) Quantification of the area of GFP-positive clones with control, MPC expression (MPC+), Got2 knockdown ( Got2 -KD), Got2 knockdown with MPC expression (MPC+, Got2 -KD), Got2 overexpression ( Got2 +), and Got2 overexpression with MPC expression (MPC+, Got2 +). Data is presented as mean ± s.d. of five biological replicates, with each 20 clonal cells analyzed for each of the specified genetic manipulations. ( k ) NADH/NAD + ratio in cells treated with 10 µM UK5099, 1 µM NMN, or 5 mM aspartate. Data is presented as mean ± s.d. of three biological replicates. ( l ) Western blot analysis of puromycin-labeled (20 µg/ml puromycin for 30 min) nascent protein in EV or MPC+ HepG2 cells cultured with 10 µM UK5099, 1 µM NMN, or 5 mM aspartate. ( m ) Quantification of intensities of puromycin labeling in EV and MPC+ cell lysates. Unpaired t -tests and one-way ANOVA tests were performed to evaluate the statistical significance of the data, and p-values are noted in the graph if significance is observed. Figure 6—source data 1. Source data related to . Figure 6—source data 2. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 6—source data 3. Original files for western blot analysis displayed in .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Plasmid Preparation, Expressing, Cell Culture, Concentration Assay, Staining, Clone Assay, Over Expression, Control, Knockdown, Western Blot, Labeling

Journal: eLife

Article Title: The fate of pyruvate dictates cell growth by modulating cellular redox potential

doi: 10.7554/eLife.103705

Figure Lengend Snippet: ( a ) Representative bright field images of empty vector (EV) or MPC over-expressing (MPC+) HepG2 spheroids cultured with either 5 mM aspartate (Asp) or 5 mM glutamate (Glu). The scale bar represents 200 µm. ( b ) Quantification of spheroid area is presented as mean ± s.d. of 30 technical replicates. ( c ) Representative bright field images of EV or MPC+ HepG2 spheroids with SLC1A3 overexpression (SLC1A3+). The scale bar represents 200 µm. ( d ) Quantification of spheroid area is presented as mean ± s.d. of 30 technical replicates. Western blots show the efficiency of MPC expression. ( e ) Quantification of d2GFP in EV or MPC+ HepG2 spheroids cultured under standard conditions or with three times the recommended concentration of non-essential amino acids cocktail (3X NEAA). qRT-PCR showing ( f ) Mpc1 , ( g ) Mpc2 , and ( h ) Got2 transcripts from fat bodies with control, MPC expression (MPC+), Got2 knockdown ( Got2 -KD), Got2 knockdown with MPC expression (MPC+, Got2 -KD), Got2 overexpression ( Got2 +), and Got2 overexpression with MPC expression (MPC+, Got2 +) normalized to rp49 transcript abundance. ( i ) Quantification of the areas of EV and MPC+ HepG2 cells and HepG2 cells GOT2 knock out (GOT2-KO) with or without MPC expression. Data is presented as mean ± s.d. of five biological replicates, with 25 randomly selected cells analyzed per replicate. Western blots show the efficiency of GOT2 knockout using the two sgRNAs described. ( j ) Representative bright field images of EV, MPC+, GOT2+, and MPC+, GOT2+ HepG2 spheroids. The scale bar represents 200 µm. ( k ) Quantification of spheroid areas is presented as mean ± s.d. of 30 technical replicates. Western blots show the efficiency of GOT2 overexpression. Unpaired t -tests and one-way ANOVA tests were performed to evaluate the statistical significance of the data, and p-values are noted in the graph if significance is observed. Figure 6—figure supplement 1—source data 1. Source data related to . Figure 6—figure supplement 1—source data 2. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 3. Original files for western blot analysis displayed in . Figure 6—figure supplement 1—source data 4. PDF files containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 5. Original files for western blot analysis displayed in .

Article Snippet: HepG2 verification was provided by ATCC and authenticated by STR profiling.

Techniques: Plasmid Preparation, Expressing, Cell Culture, Over Expression, Western Blot, Concentration Assay, Quantitative RT-PCR, Control, Knockdown, Knock-Out