Bacterial lps

Kdm5b was knocked down in THP-1 cells by transfecting siRNA specific for it. The siRNA, with the following sequence, was procured from Integrated DNA Technologies (IDT, Iowa, USA); sense: rGrCrCrArCrCrArUrUrUrGrCrArUrGrUrGrArUdTdT and antisense: rArUrCrArCrArUrGrCrArArArUrGrGrUrGrGrCdTdT. First, 2×10⁵ THP-1 cells were seeded in 24 well plates in 500μl complete RPMI medium. After 24h, 60% of the medium was removed and the cells were transfected with siRNA encapsulated in Lipofectamine^® RNAi MAX (Life Technologies, USA) at a final concentration of 5pmol following the instructions available with the transfection reagent. This was followed by addition of fresh medium equal to the volume removed before transfection. Twenty four hours after transfection, recombinant human IFNG (100ng/ml, Biolegend, USA) and bacterial LPS (1μg/ml, SIGMA, USA) were added to stimulate THP-1 cells to induce IL-12 expression. After stimulation for 24 hours, the cells were harvested and total RNA extracted for qRT-PCR determination of IL-12B and KDM5B transcripts.

Follicular B cells were purified from mouse spleen with anti-CD23-biotin (Biolegend) and streptavidin microbeads (MACS; Miltenyi Biotec). Conditional YY1 knockout in splenic B cells was performed ex vivo using TAT-CRE enzyme purified from bacteria, as previously described [33 (link), 34 (link)]. Briefly, cells were washed three times with opti-MEM (Invitrogen) and then incubated with TAT-CRE recombinant protein for 45 min at 37°C. To inactivate TAT-CRE, fetal bovine serum was added to a final concentration of 10%. Cells were washed with splenic B medium (RPMI 1640, 10% HyClone fetal bovine serum (Thermo Scientific), sodium pyruvate, 55 μM 2-mercaptoethanol (Sigma), minimal essential medium (MEM) nonessential amino acids, 2 mM l-glutamine, 1% Penicillin-Streptomycin (Invitrogen), and then cultured at 37°C in a 5% CO₂ atmosphere. Cells were activated ex vivo with 10 μg/mL bacterial LPS (Sigma) plus 20 ng/mL IL4 to stimulate proliferation and CSR.