Taqman gene probes and primers

The blood was obtained from 6 healthy donors. CD19⁺B-cells were isolated from PBMCs using EasySep Human B Cell Enrichment Kit (STEMCELL Technologies, Vancouver, BC, Canada). Purified B-cells (1x10^5 cells) were maintained in 96-well cell culture plate in RPMI 1640 medium supplemented with 10% FBS (Invitrogen), 40 ng/mL IL-4 (PeproTech Inc., NJ, USA) and 10 μg/mL CD40L (PeproTech Inc., NJ, USA) in the presence or absence of anti-human IgM+IgG (eBioscience, San Diego, CA, USA). Total RNA was extracted from cell pellets using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, MA, USA) and were incubated for 1, 6 and 24 hours. qRT-PCR reactions were performed in 384-well plates with TaqMan gene probes and primers designed by Life Technologies for TAGLN2 and three reference genes, RPS18 (assay ID: Hs01375212_g1), RPLP0 (assay ID: Hs00420895_gH) and YWHAZ (assay ID: Hs01122445_g1). These reactions were performed as described above. TAGLN2 mRNA expression was normalized to the mean of three reference genes using the 2-ΔΔCt method. Data are presented as fold change relative to expression levels of non-stimulated controls.

Peripheral blood was obtained from consenting 17 SLE patients and 12 healthy donors. Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood using Lymphocyte Separation Solution (Nakalai Tesque, Kyoto, Japan). CD19⁺B-cells were isolated from PBMCs using MACS Pan B Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Subpopulation of CD19⁺CD27⁺ (BD Pharmingen, Tokyo, Japan) memory B-cells were sorted using a flow cytometer (FACSAria, BD Biosciences, San Jose, CA). CD38 expression (BD Pharmingen) as a B-cell activation marker was examined in CD19⁺ B-cells and CD19⁺CD27⁺ memory B-cells. cDNA was synthesized using SuperScript III 1st strand cDNA Synthesis System for reverse transcription-PCR (Life Technologies, CA, USA). Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl). These reactions were performed on an Applied Biosystems ViiA 7 real time PCR system with the TaqMan Fast Advanced Master Mix (Life Technologies, CA, USA). mRNA expression was normalized to ACTB, using the 2-ΔΔCt method.

cDNA was synthesized using the SuperScript III First-Strand cDNA Synthesis System for RT-PCR (Life Technologies, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed in 384-well plates with TaqMan gene probes and primers designed by Life Technologies for MZB1 (assay ID: Hs00414907_ml) and beta-actin (assay ID: Hs01060665_gl). These reactions were performed using the ViiA 7 Real-Time PCR System (Applied Biosystems, ThermoFisher, Tokyo, Japan) with TaqMan Fast Advanced Master Mix (Life Technologies). MZB1 mRNA expression was normalized to that of beta-actin using the 2^–∆∆Ct method.