Journal: Nature communications

Article Title: PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis.

doi: 10.1038/s41467-023-38233-x

Figure Lengend Snippet: Fig. 2 | PPAR-γ mediates the high glycolytic activity of TH9 cells. a Intermediates and enzymes (red) of aerobic glycolysis. b RNA-seq of TH9 clones incubated in presence of GW9662 for 48 h and activated by αCD3/CD2/CD28 for 12 h. c Maximal glycolytic capacity of in vitro primed TH cells in the resting state and 24 h after activation with αCD3/CD2/CD28. d ECAR measurements of in vitro primed TH9 cells cultured in media with glucose of different levels and GW9662 for 48 h and activated by injection of glucose and αCD3/CD2/CD28. e Maximal glycolytic capacity of in vitro primed TH9 cells from d. f Glucose uptake by in vitro primed TH cells measured with fluorescent 2-NBDG uptake by flow cytometry at day 7. g Glucose uptake by naive TH cells primed under TH9 conditions for 7 days in presence of GW9662 or transfected with PPARG and control siRNA, respectively. Efficiency of knockdown was determined by measuring PPARG levels after trans- fection by RT-qPCR (right). h Glucose uptake of in vivo primed effector memory TH cells (TEM) sorted by flow cytometry into TH1, TH2, and TH9 cells according to their

Article Snippet: For gene silencing of SLC16A1 by siRNA, in vivo primed TH9 clones were electroporated (4D-Nucleofector, Lonza: Buffer P3, pulse E0-115) for transfection with (i) TM Select Negative Control No. 1 siRNA (Thermo Fisher Scientific), (ii) two SilencerTM Select SLC16A1 siRNAs (Thermo Fisher Scientific #s579, #s580) or (iii) MCT1 siRNA (Santa Cruz #sc-37235).

Techniques: Activity Assay, RNA Sequencing, Clone Assay, Incubation, In Vitro, Activation Assay, Cell Culture, Injection, Cytometry, Transfection, Control, Knockdown, Quantitative RT-PCR, In Vivo

Journal: Nature communications

Article Title: PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis.

doi: 10.1038/s41467-023-38233-x

Figure Lengend Snippet: Fig. 6 | IL-9 promotes T-cell proliferation in the high-glucose environment of allergic contact dermatitis. a Proliferation of in vivo primed IL-9R+ TH clones in presence of IL-9 and different concentrations of IL-2, measured with CFSE dilution by flow cytometry after 3 days. b Proliferation of in vivo primed IL-9R+ TH clones in presence of IL-9 and MCT1 inhibitor (MCT1-i) BAY-8002 measured as in a. c In vivo primed IL-9R+ TH clones were cultured in media containing glucose of different levels for 7 days. Proliferation was measured in the presence of IL-9 with CFSE dilution by flow cytometry as in a. d Glucose concentrations measured with the Glucose-GloTM Assay (Promega) of interstitial fluids of lesional skin of positive patch

Article Snippet: For gene silencing of SLC16A1 by siRNA, in vivo primed TH9 clones were electroporated (4D-Nucleofector, Lonza: Buffer P3, pulse E0-115) for transfection with (i) TM Select Negative Control No. 1 siRNA (Thermo Fisher Scientific), (ii) two SilencerTM Select SLC16A1 siRNAs (Thermo Fisher Scientific #s579, #s580) or (iii) MCT1 siRNA (Santa Cruz #sc-37235).

Techniques: In Vivo, Clone Assay, Cytometry, Cell Culture

Journal: Cancer & Metabolism

Article Title: 3-Bromopyruvate-mediated MCT1-dependent metabolic perturbation sensitizes triple negative breast cancer cells to ionizing radiation

doi: 10.1186/s40170-021-00273-6

Figure Lengend Snippet: Differential cytotoxicity and uptake of 3-bromopyruvate (3BP) in breast cancer cells. ( A ) Western blot and ( B ) the relative quantification of the optical density of each band showing the expression of MCT1 in triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, BT20, and BT549. Remaining metabolic activity (%) at ( C ) 1 or ( D ) 24 h as a measure of cell viability. Cells were treated with a concentration range of 3BP (0-300 μM) and cell viability measured using the MTT assay ( n = 4). Trypan blue exclusion after 3BP treatment (20, 100, or 150 μM) for ( E ) 1 or ( F ) 24 h was used as a metabolism-independent metric of cell viability for the measurement of the sensitivity of MDA-MB-231 and BT20 cells to 3BP. ( G ) Transfection efficiency was assessed by Western blot analysis 1 and 2 days after addition of 3BP. ( H ) BT20 cells transfected with scramble (control) siRNA (sc-BT20) or siRNA targeting MCT1 (siMCT1-BT20) were exposed to 3BP for 24 h and their viability measured ( n = 4). ( I ) Cell viability following 24 h exposure of BT20 and MDA-MB-231 cells to α-cyano-4-hydroxycinnamate (CHC), an MCT1 inhibitor, and 3BP (150 μM). Data were normalized to untreated controls. ( J ) Bromide level ( 78 Br and 80 Br) normalized to control measured by LC-MS/MS in MDA-MB-231 and BT20 cell lysates ( n = 6). Error bars represent SD, ** and * represent P < 0.05 and 0.01 respectively as determined by multiple t tests

Article Snippet: Cells were incubated overnight with siRNA against MCT1 (sc-37235) or control siRNA (sc-37007) (Santa Cruz Biotechnology, CA, USA).

Techniques: Western Blot, Quantitative Proteomics, Expressing, Activity Assay, Concentration Assay, MTT Assay, Transfection, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Cancer & Metabolism

Article Title: 3-Bromopyruvate-mediated MCT1-dependent metabolic perturbation sensitizes triple negative breast cancer cells to ionizing radiation

doi: 10.1186/s40170-021-00273-6

Figure Lengend Snippet: Colony formation assay showing the effects of 3-bromopyruvate (3BP) and ionizing radiation on triple-negative breast cancer cells. ( A ) MCT1-positive BT20 cells, ( B ) MCT1-negative MDA-MB-231 cells, and ( C ) MCT1-knockdown BT20 cells (siMCT1-BT20) were treated with a 50, 100, or 150 μM of 3BP for 1 h, a range of doses of ionizing radiation, or a combination of both where irradiation followed the 3BP treatment. siMCT1-BT20 cells were treated with 3BP and/or radiation and seeded at clonogenic density within 48-h post-transfection. For all groups, n = 6. Statistical significance was assessed with unmatched one-way ANOVA, corrected for multiple comparisons by the Tukey test. Multiplicity adjusted P value is reported. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unless otherwise marked, star symbols refer to comparisons of each treatment group with the control. Error bars show the standard deviation from the mean

Article Snippet: Cells were incubated overnight with siRNA against MCT1 (sc-37235) or control siRNA (sc-37007) (Santa Cruz Biotechnology, CA, USA).

Techniques: Colony Assay, Knockdown, Irradiation, Transfection, Control, Standard Deviation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Lactate Efflux From Intervertebral Disc Cells Is Required for Maintenance of Spine Health.

doi: 10.1002/jbmr.3908

Figure Lengend Snippet: Figure 8: MCT4 Expression is Hypoxia-inducible and HIF-dependent A-C) qRT-PCR analysis of SLC16A1 (A), SLC16A3 (B), and BSG (C) mRNA expression in rat NP cells cultured under hypoxia (1% O2) for up to 72 h. D) Representative Western blot analysis of MCT1, MCT4, and CD147 protein expression in rat NP cells cultured under hypoxia for up to 72 h. E-E’’) Densitometric analysis of MCT1 (E), MCT4 (E’), and CD147 (E’’) from Western blot experiment shown in (D). F) Representative Western blot analysis of HIF-1α, MCT1, MCT4, and CD147 protein expression in rat NP cells after silencing HIF-1α with two independent shRNAs. G-G’’) Densitometric analysis of Western blot showed in (F) above. (n=4 independent experiments). Statistical analysis: One-way ANOVA. n.s.= not significant; *, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001.

Article Snippet: Immunohistochemistry confirmed and localized expression of MCT1 (1:10; Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-50324) MCT4 (1:10; Santa Cruz Biotechnology; sc50329), and EMMPRIN/CD147 (C-19) (1:10; Santa Cruz Biotechnology; sc-9754) and qRT-PCR confirmed mRNA expression of SLC16A1, SLC16A3, and BSG in 30 human IVDs: two PM and 28 surgical samples.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Cancers

Article Title: MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma

doi: 10.3390/cancers13143468

Figure Lengend Snippet: MCT1 is over-expressed in GBM patients and associates with poor prognosis. ( A ) MCT1 expression level in 10 unmatched normal brains, 27 lower-grade gliomas (LGG), and 572 glioblastomas (GBM) patients from TCGA; *** p < 0.001 and **** p < 0.0001. ( B – I ) Kaplan-Meier survival curves of MCT1 -low and MCT1 -high GBM patients derived from microarray data from ( B ) TCGA dataset, n = 488, median OS 17.6 vs. 13.9 months, low vs. high MCT1 expression, respectively; p = 0.034; (C) REMBRANDT dataset, n = 178, median OS 541 vs. 390 days ( p = 0.018); ( D ) Ducray dataset, n = 52, median OS 16.5 vs. 13.7 months ( p = 0.043); ( E ) Lee Y dataset, n = 191, median OS 16.6 vs. 12.2 months ( p = 0.018); ( F ) Murat dataset, n = 80, median OS 20.9 vs. 14.4 months ( p = 0.003); ( G ) Gravendeel dataset, n = 155, median OS 13.3 vs. 7.8 months ( p = 0.000031); ( H ) Joo dataset, n = 54, median OS 28.4 vs. 16.9 months ( p = 0.022); ( I ) Nutt dataset, n = 28, median OS 38.2 vs. 9.1 months ( p = 0.004); and ( J ) Forest plot of hazard ratios (HR), demonstrating the relationship between MCT1 expression and GBM patients’ overall survival. The HR for each cohort is represented by a black square (its size represents the weight of the study for the meta-analysis) and 95% confidence intervals (CI) by the extending lines. The estimated pooled effect is represented by a red diamond (HR = 1.670; 95% CI, 1.434–1.945; p < 0.0001).

Article Snippet: For the generation of U251 cells stably expressing shMCT1, a pool of target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs to MCT1 knockdown (sc-37235-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Derivative Assay, Microarray

Journal: Cancers

Article Title: MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma

doi: 10.3390/cancers13143468

Figure Lengend Snippet: Cox multivariable survival analysis in GBM patients from TCGA.

Article Snippet: For the generation of U251 cells stably expressing shMCT1, a pool of target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs to MCT1 knockdown (sc-37235-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Biomarker Discovery

Journal: Cancers

Article Title: MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma

doi: 10.3390/cancers13143468

Figure Lengend Snippet: Effect of MCT1 downregulation on cell metabolism. Expression of MCT1, MCT4, HKII and HIF-1α in U251 shMCT1 cells by ( A ) Western Blot and ( B ) Immunofluorescence; Western blot MW: HIF-1α 100 kDa, HKII 100 kDa, MCT1 50 kDa, MCT4 44 kDa and β-Actin 42 kDa; Representative pictures was taken at 400× magnification. ( C ) Glucose consumption and lactate production in MCT1 knockdown cells, up to 48 h; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 U251 shCTRL vs. U251 shMCT1. ( D ) Glucose consumption and lactate production in MCT1 knockdown cells treated with CHC, up to 48 h; * p < 0.05 U251 shCTRL vs. U251 shCTRL + CHC ( E ) Glucose consumption and lactate production in MCT1 knockdown cells treated with ARC155858, up to 48 h; ** p < 0.01; *** p < 0.001 U251 shCTRL vs. U251 shCTRL + ARC155858. Results are representative of three independent experiments, each in triplicate.

Article Snippet: For the generation of U251 cells stably expressing shMCT1, a pool of target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs to MCT1 knockdown (sc-37235-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Western Blot, Immunofluorescence, Knockdown

Journal: Cancers

Article Title: MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma

doi: 10.3390/cancers13143468

Figure Lengend Snippet: MCT1 downregulation in glioma cell growth, CHC and AR-C155858 response. ( A ) Cell growth of U251 shCTRL and U251 shMCT1 over time. ( B ) Response of U251 shMCT1 cells to CHC by cell viability assay; IC50 values mean are representative of three independent experiments. ( C ) Response of U251 shMCT1 cells to ARC155858 by cell viability assay. Results are representative of three independent experiments, each in triplicate; ** p < 0.01; **** p < 0.0001 U251 shCTRL vs. U251 shMCT1.

Article Snippet: For the generation of U251 cells stably expressing shMCT1, a pool of target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs to MCT1 knockdown (sc-37235-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Viability Assay

Journal: Cancers

Article Title: MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma

doi: 10.3390/cancers13143468

Figure Lengend Snippet: MCT1 inhibition effect on glioma cells TMZ response. ( A ) Response to TMZ in U251 shCTRL and U251 shMCT1; Effect of 300 nM AR-C155858 in TMZ response for U251 shCTRL ( B ) and U251 shMCT1 ( C ). IC50 values mean are representative of three independent experiments.

Article Snippet: For the generation of U251 cells stably expressing shMCT1, a pool of target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs to MCT1 knockdown (sc-37235-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Inhibition

Journal: Cancers

Article Title: MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma

doi: 10.3390/cancers13143468

Figure Lengend Snippet: In vivo effect of MCT1 knockdown in GBMs overall survival. ( A ) Kaplan-Meier survival curves for in vivo orthotopic intracranial GBM model. Log-rank test shMCT1 vs. shCTRL vs. shMCT1+ TMZ vs. shCTRL+ TMZ, p < 0.0001 n = 11; n = 11; n = 5; n = 5 per group, respectively; ( B ) Hematoxylin-eosin staining of NSG mice brains; Representative pictures of immunohistochemistry for Ki67 in brain tissues of NSG mice at 400× magnification. ( C ) MCT1, MCT4 and CAIX expression in shCTRL and shMCT1 plus TMZ in brain tissues at 400× magnification.

Article Snippet: For the generation of U251 cells stably expressing shMCT1, a pool of target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs to MCT1 knockdown (sc-37235-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: In Vivo, Knockdown, Staining, Immunohistochemistry, Expressing