peredox nadh nad sensor  (Addgene inc)

Journal: Nature Communications

Article Title: Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms

doi: 10.1038/s41467-024-51117-y

Figure Lengend Snippet: a Heatmap showing the expression of YAP target genes in P and Q MEFs as determined by RNA sequencing. Data represent values from three independent experiments, and Z-scored log2-fold change values are color-coded as indicated. b , c Localization analysis of YAP (green) in control or E-cadherin KO MEFs in the Q state by immunofluorescence. Hoechst 33342 (blue) was used to show the nuclei, and the scale bar size represents 10 μm. Each experiment included observation of at least 10 randomly selected fields (400× magnification). The quantified percentages (%) of nuclear (red) and cytosolic (green) YAP are indicated, and the data represent the mean ± SEM. The statistical significance of the differences was determined by two-way ANOVA; P < 0.0001. d Luciferase assays with the 8 × GTIIC-Lux reporter in control or E-cadherin KO MEFs in the P and Q states. Data are normalized to sgCdh1 (-) P cells (wild-type) and are presented as the mean ± SEM ( n = 3–5). P values by unpaired two-tailed student’s t -test are indicated except for ns (P E-cad KO P = 0.9571, Q E-cad KO P = 0.0713). e Luciferase assays with the 8 × GTIIC-Lux reporter in control or TEAD1 KO MEFs in the P and Q states. Data are normalized to sgTead1 (-) P cells (wild-type) and are presented as the mean ± SEM of n = 5 biologically independent samples. **** P < 0.0001, ns P = 0.1242 using unpaired two-tailed t -test. f Glycolytic function monitored by extracellular acidification rate (ECAR) in control or TEAD1 KO MEFs in the P and Q states. Data are presented as the mean ± SEM of n ≥ 9 biologically independent samples. **** P < 0.0001, ns P = 0.0744 using unpaired two-tailed t -test. g Immunoblots and protein quantification of TEAD1, glycolytic enzymes, and p27 kip1 in control or TEAD1 KO MEFs in the P and Q states. Each immunoblot is a representative of four biologically independent experiments. Values are the mean ± SEM of 3–4 biologically independent experiments. P values by unpaired two-tailed student’s t -test are indicated except for **** P < 0.0001 and ns (PFK1 P = 0.4610, PKM2 P = 0.1934, P p27 kip1 P = 0.1074, Q p27 kip1 P = 0.7180). HK hexokinase, PFK1 phosphofructokinase 1, PKM2 pyruvate kinase M2. Values are the mean ± SEM of 3–4 biologically independent experiments.

Article Snippet: For cytosolic NAD + /NADH ratio measurement with the Peredox probe, cells were transduced with the pMOS023: Peredox NADH/NAD + sensor (cytosolic) plasmid (Addgene 163060).

Techniques: Expressing, RNA Sequencing, Control, Immunofluorescence, Luciferase, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms

doi: 10.1038/s41467-024-51117-y

Figure Lengend Snippet: a Whole-cell NAD + /NADH ratio of P and Q MEFs. Data are presented as the mean ± SEM of 6 biologically independent samples. **** P < 0.0001 using unpaired two-tailed t -test. b Cytosolic NAD + /NADH ratio of P and Q MEFs measured by the pMOS023: Peredox NADH/NAD + sensor (cytosolic) using flow cytometry. The mean MFI was calculated based on 4 biologically independent experiments. Data are presented as the mean ± SEM. **** P < 0.0001 using unpaired two-tailed t -test. c The relative abundance of mitochondrial NADH in P and Q MEFs measured by the pC1-mitoRexYFP sensor using flow cytometry. The mean MFI was calculated based on 4 biologically independent experiments. Data are presented as the mean ± SEM. **** P < 0.0001 using unpaired two-tailed t -test. d Immunoblots and protein quantification of MDH1 and MDH2 in P and Q MEFs. Values are the mean ± SEM of 4 biologically independent experiments. **** P < 0.0001, ns P = 0.3892 using unpaired two-tailed t -test. e The whole-cell NAD + /NADH ratio in either empty vector (EV), cytosolic (Cyto) or mitochondrial (Mito) LbNOX-expressing P or Q MEFs. Representative immunoblots confirm Flag-tagged LbNOX expression. Data are presented as the mean ± SEM of 4-6 independent experiments. P value by unpaired two-tailed student’s t -test is indicated except for **** P < 0.0001. f Glycolysis measured by the extracellular acidification rate (ECAR) in EV-, CytoLbNOX-, and MitoLbNOX-expressing cells in P and Q MEFs. Data are presented as the mean ± SEM of 3–4 biologically independent samples. P value by unpaired two-tailed student’s t -test is indicated except for ns (P MitoLbNOX P = 0.5999, Q CytoLbNOX P = 0.3209, Q MitoLbNOX P = 0.5020). g Basal respiration measured by oxygen consumption rate (OCR) of P and Q MEFs cultured in vehicle or 0.2 μM rotenone for 24 h. Values are the mean ± SEM of 4–5 independent experiments. ****p < 0.0001 using unpaired two-tailed t -test. h The whole-cell NAD + /NADH ratio in P or Q MEFs cultured in vehicle or 0.2 μM rotenone for 24 h. Data are presented as the mean ± SEM of 4 biologically independent samples. **** P < 0.0001 using unpaired two-tailed t -test. i The cytosolic NAD + /NADH ratio in P or Q MEFs cultured in vehicle or 0.2 μM rotenone for 24 h. Data are presented as the mean ± SEM of 6 biologically independent samples. **** P < 0.0001 using unpaired two-tailed t -test. j Glycolysis measured by the extracellular acidification rate (ECAR) of P and Q MEFs cultured in vehicle or 0.2 μM rotenone for 24 h. Data are presented as the mean ± SEM of 6–8 biologically independent samples. **** P < 0.0001 using unpaired two-tailed t -test. k Simplified schematic of the 13 C labeling patterns of metabolites in glycolysis and the TCA cycle with [U- 13 C] glucose tracing (Created with Biorender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license). Red fills indicate 13 C-labeled carbons. The mitochondrial pyruvate carrier (MPC) and Complex I are depicted on the mitochondrial membrane. l Relative ratio of (M + 3) lactate and pyruvate after 25 mM [U- 13 C] glucose labeling in Q MEFs cultured with vehicle or 0.2 μM rotenone for 24 h. Means ± SEMs ( n = 3) are shown. P value by unpaired two-tailed student’s t -test is indicated. m Relative abundance of intracellular (M + 2) citrate after 25 mM [U- 13 C] glucose labeling in Q MEFs cultured with vehicle or 0.2 μM rotenone for 24 h. Means ± SEMs ( n = 3) are shown. P value by unpaired two-tailed student’s t -test is indicated.

Article Snippet: For cytosolic NAD + /NADH ratio measurement with the Peredox probe, cells were transduced with the pMOS023: Peredox NADH/NAD + sensor (cytosolic) plasmid (Addgene 163060).

Techniques: Two Tailed Test, Flow Cytometry, Western Blot, Plasmid Preparation, Expressing, Cell Culture, Labeling, Membrane

Journal: Nature Communications

Article Title: Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms

doi: 10.1038/s41467-024-51117-y

Figure Lengend Snippet: a Immunoblots and protein quantification of SLC25A11 and SLC25A13 in P and Q MEFs. Vinculin was monitored as a loading control. Representative immunoblot is shown. Values are the mean ± SEM of 4 biologically independent experiments. P values by unpaired two-tailed student’s t -test are indicated. b The schematic of malate-aspartate shuttle (MAS) (created with Biorender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license). MDH malate dehydrogenase, GOT glutamic-oxaloacetic transaminase, SLC25A11 solute carrier family 25 member 11. c The cytosolic NAD + /NADH ratio of P and Q MEFs cultured in vehicle or 2 mM AOA for 24 h measured by the pMOS023: Peredox NADH/NAD + sensor (cytosolic) using flow cytometry. Values are the mean ± SEM of 6 biologically independent experiments. **** P < 0.0001 and ns P = 0.4623 using unpaired two-tailed t -test. d Immunoblot and protein quantification of SLC25A11 in MEFs. α-Tubulin was monitored as a loading control. Each immunoblot is representative of 6 biologically independent experiments. Values are the mean ± SEM of 6 biologically independent experiments; **** P < 0.0001 using unpaired two-tailed t -test. e The cytosolic NAD + /NADH ratio of P and Q MEFs with or without SLC25A11 deletion. The ratios are measured by the pMOS023: Peredox NADH/NAD + sensor (cytosolic) using flow cytometry. Values are the mean ± SEM of 4 biologically independent experiments. **** P < 0.0001, ns P = 0.2711 using unpaired two-tailed t -test. f Proliferation rates of P MEF cells cultured in vehicle or 2 mM AOA or AOA + 4 mM methyl pyruvate for 24 h. The relative ratio of doublings per day is presented as the mean ± SEM of 4 biologically independent samples. **** P < 0.0001, ns P = 0.0605 using unpaired two-tailed t -test. g The cytosolic NAD + /NADH ratio of P cells cultured in vehicle or 2 mM AOA or AOA + 4 mM methyl pyruvate for 24 h. Data are presented as the mean ± SEM of 4 biologically independent samples. P value by unpaired two-tailed student’s t -test is indicated except for **** P < 0.0001. h Glycolysis measured by the extracellular acidification rate (ECAR) in P cells cultured in vehicle or 2 mM AOA or AOA + 4 mM methyl pyruvate for 24 h. Data are presented as the mean ± SEM of 5 biologically independent samples. P values by unpaired two-tailed student’s t -test are indicated except for ns P = 0.4414. i The cytosolic NAD + /NADH ratio of P MEFs with or without SLC25A11 deletion and cultured in vehicle or 4 mM methyl pyruvate for 24 h. Data are presented as the mean ± SEM of 4 biologically independent samples. **** P < 0.0001 using unpaired two-tailed t -test.

Article Snippet: For cytosolic NAD + /NADH ratio measurement with the Peredox probe, cells were transduced with the pMOS023: Peredox NADH/NAD + sensor (cytosolic) plasmid (Addgene 163060).

Techniques: Western Blot, Control, Two Tailed Test, Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

doi: 10.1038/s41598-021-81349-7

Figure Lengend Snippet: Metformin increases glycolysis in intestinal cells. ( a ) Changes in extracellular acidification rate (ECAR) during the glycolytic stress test wherein intestinal cells were pre-treated with control or 1 mM metformin for 24 h. Results were normalised to the protein concentration measured by BCA assay. Dotted lines indicate when compound were added: Gluc glucose (10 mM), OliA oligomycin-A (1 μM), 2-DG 2-deoxyglucose (50 mM). Inset: Effect of metformin on measured parameters of the glycolysis stress test. n = 10 wells from 3 independent experiments. NS not significant. ***P < 0.001. Two-way ANOVA and Bonferroni post-hoc test. ( b ) Example traces of Perceval fluorescence ratios from Control and Metformin treated cells in response to 2-DG (50 mM), with violin plots shows of the change in fluorescent intensity ratio (FI). ***P < 0.001, Student’s t test. Control; n = 14 from 4 dishes and metformin; n = 17 from 4 dishes. ( c ) Schematic of glycolysis and effects of inhibitors. ( d ) Effects of AR-C155858 (AR-C, 10 μM) and Syrosingopine (Syro, 50 μM) on Perceval fluorescence in Control (red) and Metformin (blue) treated cells. 10 mM glucose (Gluc) was present throughout, and 2-DG (50 mM) added as indicated. Error bars are mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni post-hoc test. Control: n = 22 cells from 6 dishes; metformin: n = 16 cells from 5 dishes. Error bars are mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni post-hoc test. ( e ) As ( d ), for oxamate (50 mM). Control: n = 23 cells from 6 dishes; metformin: n = 25 cells from 6 dishes. ( f ) Supernatant lactate levels in control (red) and metformin (blue) pre-treated cultures in glucose (10 mM, 10G), with oxamate (50 mM) or AR-C, (10 μM) and Syro (50 μM). Error bars are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs control. ††† P < 0.001 vs pre-treated conditions in 10G. Two-way ANOVA and Bonferroni post-hoc test. n = 2–3 wells from 4 independent experiments except AR–C/Syro (3 independent experiments). ( g ) Effects of oxamate on Peredox fluorescence in control (red) and metformin (blue) treated cells. Glucose (Gluc, 10 mM) and oxamate (50 mM) were applied as indicated. Error bars are median ± 95% confidence intervals. *P < 0.05, Mann–Whitney test. Control: n = 15 cells from 4 dishes; Metformin: n = 18 cells from 4 dishes.

Article Snippet: The T-Sapphire and mCitrine fluorescence of the Peredox sensor (32386, Addgene) was sequentially excited at 405 ± 20 nm and 480 ± 10 nm respectively, and images were acquired every 10 s. T-Sapphire fluorescence (emitted from excitation at 405 ± 20 nm) was increased correlating with cytosolic NADH concentrations, whilst the mCitrine fluorescence remained constant throughout the experiment.

Techniques: Control, Protein Concentration, BIA-KA, Fluorescence, MANN-WHITNEY