Ethics committee approval
Care, use, and treatment of mice in this study were in strict agreement with international guidelines for the care and use of laboratory animals. This study was approved by the Animal Ethics Committee of Beijing Institute of Basic Medical Sciences.
Mice
Seven-to-nine-week-old female or male C57BL/6 and Balb/c mice were purchased from the Chinese Academy of Medical Sciences (Beijing, China). Seven-to-nine-week-old female lupus-prone MRL/MpJ/lpr/lpr (MRL/lpr) mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China) and described previously [20]. EAE induction on C57BL/6 mice was performed, as previously described [10]. All mice were bred in our animal facilities under specific pathogen-free conditions.
Treatment of lupus-prone mice with TACI-IgG
Treatment of lupus-prone mice with TACI-IgG was previously described [10]. Simply, Lupus-prone MRL/lpr mice were divided into the following two groups: 1, TACI-IgG treated; 2, control IgG-treated. Six lupus-prone mice per group were injected i.p. with 5 mg/kg TACI-IgG and control IgG (Rongchang pharmaceuticals, LTD, Shandong province, China) at 1, 2, 3, and 4 week (twice per week) after the mice reached 6 month of age.
Cell sorting
The splenocytes were separated from three 7–9-week female C57BL/6 mice per group, stained with anti-mouse CD5 (clone no. 53-7.3) and B220 (clone no. RA3-6B2, eBioscience, USA) antibodies. CD5+ and CD5− B220+B cells were sorted by flow cytometry (FACS). All flow cytometry data were acquired with FACSCanto, FACSCantoII, or FACSAria (BD Biosciences), gated on live lymphocyte-sized cells on the basis of forward and side scatter, and analyzed using FlowJo software (Tree Star, Ashland, OR). CD19+ and B220+ B cells were separated by CD19 (Cat No. 130-052-201) and B220 (Cat No. 130-049-501, Miltenyi Biotec, Germany) Microbeads, respectively.
Cell culture and transfection
Mouse myeloma cell line SP 2/0 cells and human embryonic kidney HEK 293T cells were from the American Type Culture Collection (ATCC; Rockville, MD, usa) and described previously [21]. All cells were maintained in complete RPMI 1640 medium in a humidified 5% CO2 atmosphere at 37 °C. For cell transfection, BC094916 cDNA (General Biosystems, Anhui, China) was cloned into lentiviral vector LV201 or LV122 (Fugene Corp., Guangzhou, China) to generate BC094916 and BC094916-EGFP fusion protein, respectively. BC094916-expressing LV201 or LV122 was then transfected into SP 2/0 cells, and stable transfectants were identified by drug selection (Puromycin, Sigma, 10 μg/ml).
B cells were stimulated in vitro with LPS
B cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 50 mM 2-mercaptoethanol. Cells were stimulated with 10 μg/ml LPS (Sigma L2630 from Escherichia coli 0111:B4; Sigma, St Louis, MO).
Affymetrix microarrays
Affymetrix microarrays were performed by GMINIX Ltd (Shanghai, China) and described previously [22]. Total RNA was extracted from B cells with Trizol and purified over Qiagen RNeasy columns (Qiagen). Synthesis and labeling of RNA and hybridization of arrays were conducted. Stained arrays (430 2.0) were scanned on an Agilent Gene Array Scanner (Affymetrix).
RNA sequencing
RNA was isolated from cells with Qiagen RNeasy Micro or Mini Kits (on the basis of cell number), according to the manufacturer’s instructions. RNA-seq was done with an Illumina HiSeq2500 instrument at Genewiz corp., Suzhou, China.
Immunofluorescence and confocal microscopy
Cells were observed under a fluorescence microscope or seeded onto glass coverslips in 24-well plates, washed with PBS, fixed in 4% formaldehyde solution and permeabilized with 0.2% Triton X-100/PBS. Cells were blocked with 2% BSA in PBS for 30 min. Coverslips were incubated with DAPI and observed under a confocal laser scanning microscope.
Measure of cell proliferation with cell counting kit-8 (CCK8) assay
Measure of cell proliferation with cell counting kit-8 (CCK8) assay was described previously [23]. CCK8 kit was purchased from Dojindo Molecular Technologies, Inc. Rockcille, MD, USA. Briefly, 100 μl of cell suspension (5000 cells/well) in a 96-well plate were cultured for an appropriate length of time (e.g., 0, 1 or 2 days) in a humidified incubator (e.g., at 37 °C, 5% CO2). 10 μl of CCK-8 solution was added to each well of the plate and the plate was incubated for 1–4 h in the incubator. Measure the absorbance at 450 nm using a microplate reader.
Quantitative PCR analysis
Quantitative PCR analysis has been described in our previous studies [22, 24]. Briefly, total RNA was extracted from B cells with Trizol (Invitrogen Life Technologies). The final RNA pellets were dissolved in 0.1 mM EDTA (2 μl/mg original wet weight). Reverse transcription reactions were carried out on 22 μl of sample using superscript II RNAse H-Reverse Transcriptase (Invitrogen Life Technologies) in a reaction volume of 40 μl. All samples were diluted in 160 μl nuclease-free water. qPCR was employed to quantify mouse gene expression from the cDNA samples. Mouse gene expression was normalized to the levels of the β-actin gene.
Immunoblot analysis
Whole-cell lysates were prepared for Western blotting. Twenty-five micrograms of cell protein were electrophoretically separated on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane, which was then blocked by incubation for 1 h at room temperature in 5% fat-free dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). The blots were then incubated overnight at 4 °C with rabbit antibodies against anti-mouse GAPDH (KM9002, SunGene Biotech), Xbp-1 (ab37152, Abcam), blimp1 (sc-25380, Santa Cruz Biotech), Bcl2 (ab59348, Abcam), Bcl6 (sc-858, Santa Cruz Biotech), Aid (sc-25620, Santa Cruz Biotech), Myc (66004-1-lg, Proteintech), p53 (#2524, Cell Signaling Tech), Mcl1 (ab32087, Abcam), EGFP (ab290, Abcam) antibodies diluted 1:1000 in TBS-T containing 5% bovine serum albumin, washed for 25 min with TBS-T, and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary F(ab’)2 (Zymed Laboratories, San Francisco, CA) (1:20 000 in TBS-T containing 5% bovine serum albumin), then bound antibody was visualized using the ECL detection system (Amersham, Arlington Heights, IL).
Propidium iodide (PI)/FACS (cell cycle) analysis
Cell cycle analysis was described previously [22]. Cells were collected and washed 1 time with 5–10 ml of 1× PBS. Cells were suspended in 500 μl 1× PBS containing + 0.1% Glucose (at 4 °C) and 5 ml of cold 70% EtOH (kept at −20 °C) was immediately added, mixed, and kept at 4 °C for 1 h. Cell were then spun down and washed once with 1 × PBS (10 ml). Without adding more PBS, cells were then spun again for 2 min so that the residual PBS could be removed and cells were then suspended in 300 μl 69 μM propidium iodide (Cat# 537059, Calbiochem, San Diego, CA) solution with 38 mM Na Citrate (Cat# C7254 Sigma, St. Louis, MO) and 20 μl of 10 mg/ml RNase (Cat# R4875, Calbiochem, San Diego, CA). Cells were mixed, incubated at 37 °C for 30–45 min and analyzed by FACS.
Annexin V/PI staining
Apoptosis detection kit was purchased from Sungenebiotech, Tianjing, China. Cells were centrifuged at 335×g for 10 min and resuspended in 2 ml 1× phosphate buffered saline (PBS) (no calcium, no magnesium). Cells were centrifuged at 335×g for 10 min and resuspended in 1 ml 1X Annexin V binding buffer. A total of 5 μl APC-conjugated Annexin V (Cat No. AO2001-02, Sungenebiotech, Tianjing, China) was added and the tubes incubated in the dark for 15 min at room temperature. A total of 100 μl of 1x Annexin V binding buffer was added to each reaction tube (final volume: ~ 200 μl). PI (4 μl, Cat No. AO2002-H, Sungenebiotech, Tianjing, China) was diluted 1:10 in 1x Annexin V binding buffer and a final PI concentration of 2 μg/ml was added in each sample. Tubes were incubated in the dark for 15 min at room temperature. 1× Annexin V binding buffer (500 μ) was added to wash the cells. Then the samples were ready to be analyzed by flow cytometry (FACS).
SP 2/0 xenograft mouse model
To evaluate tumor growth in mouse models, 200 μl of cell suspension from 5 × 106 SP 2/0 expressing GFP (vector) or SP 2/0 cells expressing GFP and BC094916 (BC094916) were subcutaneously injected into the left and right sides of the back of each Balb/c mouse. Mice were sacrificed on day 8 after the injection. Tumor volumes were determined by measuring the major (L) and minor (W) diameters with an electronic caliper. The tumor volume was calculated according to the following formula: tumor volume = π/6 × L × W2.
Creb1 and Bcl2 promoter reporting gene analysis
Promoter reporting gene analysis has been described in our previous studies [25]. The firefly luciferase reporter plasmid pEZX-PG04.1 (Fugene Corp., Guangzhou, China) with the 5′-flanking region from start codon upstream − 1510 ~ + 173 of mouse Creb1 gene or − 1323 ~ + 160 of mouse Bcl2 gene. 0.5 μg Lv201/BC094916, 0.5 μg firefly luciferase reporter plasmids pEZX-PG04.1/Creb1 promoter or Bcl2 promoter (General Biosystems, Anhui, China), and 0.05 μg Renilla luciferase reporter vector pRL-SV-40 vector (cat# E2231, Promega Corp.) were co-transduced into 4 × 105 SP 2/0 or 293T cells in 12-well plate by using 6 μL Lipofectamine®2000 Reagent (Cat# 11668-019, Invitrogen Corp.). On day 3, sequential measurement of firefly luciferase (Reporter #1) followed by Renilla luciferase activity (Reporter #2) was assessed on 1420 Multilabel Counter (1420 Victor 3, PerkinElmer Corp.), and analyzed. The results were shown as the ratio of firefly to Renilla luciferase activity.
Statistics
Statistics were analyzed by using GraphPad Prism (version 5.0, GraphPad Software Inc., USA). The data were shown as mean ± standard error of the mean (SEM). Student’s t test was employed to determine significance between two groups (paired or unpaired) and Two-Way ANOVA analysis was used to determine significance among several groups. Differences were considered statistically significant when p < 0.05.