Scrambled sirna ctrl si

c-Maf siRNA, CD69 siRNA, STAT3 siRNA, STAT5 siRNA, or scrambled siRNA (Ctrl Si) were synthesized by Genepharma (Shanghai, China). Target sequences for c-Maf: 5′-ACCCUUCCUCUCCCGAAUUTT-3′ (sense), 5′-GGCCAUGGAAUAUGUUAAUTT-3′ (antisense); CD69: 5′-CCAUGGACCAGUAUACAUTT-3′ (sense), 5′-AUGUAUACUGGUGCCAUGGTT-3′ (antisense); STAT3: 5′-CCCGCCAAAUUAAGAATT-3′ (sense), 5′-UUCUUAAUUUGUUGGCGGGTT-3′ (antisense); STAT5: 5′-GGAUGAGAGCAUGGAUGUUTT-3′ (sense), 5′-AACAUCCAUGCUCUCAUCCTT-3′ (antisense); Scrambled sequences for c-Maf, CD69, STAT3, and STAT5: 5′-UUCUCGAACGUGUCACGUTT-3′ (sense), 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). For siRNA transfection, EL4 cells (5 × 10⁵/well) were seeded in 24-wells plates overnight and transfected using Lipofectamine^TM RNAiMAX transfection reagent for 48 h (Invitrogen).

HSF1 siRNA or scrambled siRNA (Ctrl Si) were synthesized by GenePharma (Shanghai, China). The target sequences for HSF1 are listed in Table 1. For siRNA transfection, CD4⁺ T cells (1 × 10⁶/well) were seeded in 24-well plates overnight and transfected using Lipofectamine ^TM RNAi MAX transfection reagent (Invitrogen, USA) for 24 h. For inhibition of HSF1, 1 × 10⁶ CD4⁺ T cells were seeded overnight in 48-well plates and 5 μM HSF1 inhibitor KRIBB11 (Sigma Aldrich, USA) were added for 24-48 h. DMSO was used as a negative control.
The HSF1 ORF (open reading frame) was amplified by PCR and inserted into a lentivirus vector (pPCDH-CMV-MCS-EF1-GFP) after sequencing confirmation. CD4⁺ T cells were then transfected with the packaged recombinant lentivirus or scrambled control vector. Briefly, viral supernatant was concentrated using PEG-it virus precipitation solution (System Biosciences, USA). High titer virus was then incubated with isolated CD4⁺T cells, followed by spinoculated for 2 h at 300g with polybrene (8 mg/ml) and cultured for 48 h. The expression of CD69 after transduction was confirmed by using FACS.