Fig. 5: HMB regulates HIF-1α expression in human CD4⁺ T cells.

a The protein expression of HIF-1α in TCR-stimulated CD4⁺ memory T cells treated with or without Bi2 was analyzed (n = 5). As a control for HIF-1α, PANC-1 cells were incubated for 8 h under 2% or 21% O₂. A representative immunoblot is shown (left). The band intensity in the immunoblots was quantified by densitometry, except for the PANC-1 data. β-Actin was used as a normalization control (right). b Freshly isolated human CD4⁺ memory T cells were activated and infected with GFP lentivirus containing BCAT1, HPD, or HPDL shRNA for 24 h. shRNA⁺ cells expressing GFP were sorted and cultured for another 3 days with TCR stimulation. The mRNA expression of HIF-1α was analyzed by RT‒qPCR (n = 3–5). c HIF-1α expression in human CD4⁺ memory T cells was measured 24 h after TCR stimulation with Bi2 (n = 5). The cells were supplemented with HMB (0.4 mM) for the indicated time before harvest. d Quantitative PCR analysis of HIF-1α target gene expression after 72 h of TCR stimulation in human CD4⁺ memory T cells from HCs (n = 6). HMB (0.4 mM) was used to pretreat the cells for 1 h before TCR stimulation. e IL-17A and IFN-γ production were quantified at 72 h after TCR stimulation with or without Bi2 (10 μM), VH298 (100 nM), or HMB (0.4 mM) (n = 5). f mRNA expression of HIF-1α was measured at 4 h after TCR stimulation. The cells were supplemented with HMB (0.4 mM) for the indicated time before harvest (n = 5). g CD4⁺ memory T cells were stimulated with anti-CD3/CD28-coated microbeads for 1 h in the absence or presence of Bi2. Cell lysates were prepared at the indicated times and immunoblotted with phospho-p70-S6K and total p70-S6K (n = 3 independent experiments). h CD4⁺ memory T cells were stimulated with anti-CD3/CD28 Abs for 1 h. The cells were supplemented with HMB (0.4 mM) for the indicated time before harvest. Cell lysates were prepared at 1 h poststimulation and immunoblotted for phospho-p70-S6K and total p70-S6K (n = 5 independent experiments). The graph shows the band intensity quantified by densitometry. The graphs show the means ± SEMs. *p < 0.05 and **p < 0.01 according to the Mann‒Whitney U test [a, b (left), c–f, h] or two-tailed unpaired t test [b (right)].