Journal: Cell reports

Article Title: HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

doi: 10.1016/j.celrep.2024.114103

Figure Lengend Snippet: Figure 6. Reduced cell proliferation as consequence of HIF1a-dependent lower glycolysis inhibition (A) Cell proliferation assays in 786-O-HIF1a cells and their corresponding control cells in the presence or absence of extracellular pyruvate or aKB (1 mM) (left; n = 9–10) in G55 control cells and G55 cells treated with 0.2 mM DMOG in the presence or absence of extracellular pyruvate or aKB (1 mM) (middle; n = 9) and MDA- MB-231 control cells and MDA-MB-231 cells treated with 0.5 mM DMOG in the presence or absence of extracellular pyruvate or aKB (1 mM) (right; n = 3). (B) On the left, representative western blots of the GAPDH and b-ACTIN protein in G55 control cells and GAPDH-silenced G55 cells. In the middle, cell proliferation assay in G55 control cells and GAPDH-silenced G55 cells (n = 9). On the right, the relative amount of extracellular lactate released by G55 control cells and GAPDH-silenced G55 cells, (n = 6). In the bar graphs, the values represent the mean ± SEM, and statistical analysis was performed using a two-tailed unpaired t test when comparing 2 groups, with Welch’s correction when needed, or two-way ANOVA followed by Tukey’s post hoc test: ***p < 0.001 represent significant p values.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-HIF1a BD Transduction Laboratories Cat#610959; RRID: AB_398272 Mouse monoclonal anti-CAIX Santa Cruz Biotechnology Cat#sc-365900; RRID: AB_10846466 Monoclonal anti-b-actin-peroxidase Sigma-Aldrich Cat#A3854; RRID: AB_262011 Rabbit polyclonal anti-HIF-1a (C-Term) Cayman Chemical Cat#10006421; RRID: AB_409037 Rabbit polyclonal anti- PDK1 Enzo Life Sciences, Inc. Cat#ADI-KAP-PK112; RRID: AB_10618932 Goat polyclonal anti-HSP 60 Santa Cruz Biotechnology Cat#sc-1722; RRID: AB_2233354 Rabbit polyclonal anti-GAPDH Abcam Cat#ab9485; RRID: AB_307275 Rabbit polyclonal anti-GOT1 Proteintech Cat#14886-1-AP; RRID AB_2113630 Rabbit polyclonal anti-GOT2 Sigma-Aldrich Cat#HPA018139; RRID AB_1849903 Chemicals, peptides, and recombinant proteins Doxycycline Sigma-Aldrich Cat#D9891 Dimethyloxalylglycine (DMOG) Enzo Life Sciences, Inc. Cat#BML-EI347 Metformin Sigma-Aldrich Cat#D150959 Sodium pyruvate Lonza Cat#BE13-115E Sodium a-ketobutyrate (aKB) Sigma-Aldrich Cat#K0875 L-Glutamine-5-13 C Cambridge Isotope Laboratories, Inc.Cat#CLM-1166 Protease inhibitors Sigma-Aldrich Cat#P8340 Phosphatase inhibitors Sigma-Aldrich Cat#P5726 Pyridine Sigma-Aldrich Cat#270970 Methoxyamine hydrochloride Sigma-Aldrich Cat#226904 5-3H-Glucose Perkin Elmer Cat#NET531001MC Dodecyltrimethylammonium bromide (DTAB) Sigma-Aldrich Cat#D8638 Critical commercial assays L-Lactate Assay Kit (Colorimetric/ Fluorometric) Abcam Cat#ab65330 DC protein Assay Kit Bio-Rad Cat#5000112 PierceTM BCA Protein Assay Kit Thermo Fisher Scientific Cat#23225 NAD/NADH-GloTM Assay Kit Promega Cat#G9071 Clarity Western ECL Substrate Bio-Rad Cat#1705061 Super-Signal West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific Cat#34096 Experimental models: Cell lines 786-O Provided by Dr. Marı́a J. Calzada N/A G55 Provided by Dr. Till Acker N/A MDA-MB 231 Provided by Dr. Massimiliano Mazzone N/A A549 Provided by Dr. Massimiliano Mazzone N/A Murine embryonic fibroblasts (MEFs) This study N/A Oligonucleotides Control siRNA Santa Cruz Biotechnology Cat#sc-37007 HIF1a siRNA Santa Cruz Biotechnology Catsc-35561 GAPDH siRNA Santa Cruz Biotechnology Cat#sc-35448 GOT1 siRNA Dharmacon Cat#J-011673 (Continued on next page) 14 Cell Reports 43, 114103, April 23, 2024

Techniques: Inhibition, Control, Western Blot, Proliferation Assay, Two Tailed Test

Journal: Cell reports

Article Title: HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

doi: 10.1016/j.celrep.2024.114103

Figure Lengend Snippet: Figure 6. Reduced cell proliferation as consequence of HIF1a-dependent lower glycolysis inhibition (A) Cell proliferation assays in 786-O-HIF1a cells and their corresponding control cells in the presence or absence of extracellular pyruvate or aKB (1 mM) (left; n = 9–10) in G55 control cells and G55 cells treated with 0.2 mM DMOG in the presence or absence of extracellular pyruvate or aKB (1 mM) (middle; n = 9) and MDA- MB-231 control cells and MDA-MB-231 cells treated with 0.5 mM DMOG in the presence or absence of extracellular pyruvate or aKB (1 mM) (right; n = 3). (B) On the left, representative western blots of the GAPDH and b-ACTIN protein in G55 control cells and GAPDH-silenced G55 cells. In the middle, cell proliferation assay in G55 control cells and GAPDH-silenced G55 cells (n = 9). On the right, the relative amount of extracellular lactate released by G55 control cells and GAPDH-silenced G55 cells, (n = 6). In the bar graphs, the values represent the mean ± SEM, and statistical analysis was performed using a two-tailed unpaired t test when comparing 2 groups, with Welch’s correction when needed, or two-way ANOVA followed by Tukey’s post hoc test: ***p < 0.001 represent significant p values.

Article Snippet: Temporary siRNA gene silencing was achieved using a specific HIF1a siRNA (sc-35561, Santa Cruz Biotechnology), GAPDH siRNA (sc-35448, Santa Cruz Biotechnology), GOT1 siRNA (J-011673, Dharmacon), GOT2 siRNA (J-011674) and a control siRNA (sc-37007, Santa Cruz Biotechnology).

Techniques: Inhibition, Control, Western Blot, Proliferation Assay, Two Tailed Test