Additional file 1: of Iguratimod represses B cell terminal differentiation linked with the inhibition of PKC/EGR1 axis
Creators: name: Ye, Yan
name: Liu, Mei
name: Tang, Longhai
name: Du, Fang
name: Liu, Yuanhua
name: Hao, Pei
name: Fu, Qiong
name: Guo, Qiang
name: Yan, Qingran
name: Zhang, Xiaoming
name: Bao, Chunde
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Plant Biology

description: Figure S1. Comparison of different protocols for ASC differentiation in vitro. Human B cells were sorted by CD19 beads from PBMC of healthy donors and stimulated with ten different conditions for 5 days. Frequencies of CD19+CD20-CD27hiCD38hi ASC (A) and immunoglobulins from culture supernatant (B) were shown. (C) FCM plots and (D) cumulative data of ASC generation following CpG/IL-2/IL-10 stimulation at different time points were shown (n = 3). One representative of at least three independent experiments was shown. Data were shown as mean ± SEM and analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons (C). *P &lt; 0.05, ***P &lt; 0.001, ****P &lt; 0.0001. Figure S2. Iguratimod does not affect B cell apoptosis, activation, or proliferation. Human B cells were stimulated with CpG/IL-2/IL-10, in the presence of vehicle (DMSO) or 10 μM iguratimod. (A, B) Both apoptotic (Annexin V+PI-) and dead cells (Annexin V+PI+) were not significantly changed with iguratimod treatment at 48 h (n = 3). (C) CD69 or CD25 stains were not changed at 48 h. (D) Cumulative data of the MFIs of CD69 and CD25 were shown (n = 3). (E, F) Proliferating B cell population (CSFElo) was not significantly changed at day 5 (n = 3). One representative of at least three independent experiments was shown. Data were shown as mean ± SEM (B, D, F) and analyzed by Student’s t test (B, D) or one-way ANOVA (F). MFI, median fluorescence intensity. Figure S3. Iguratimod does not affect the phosphorylation status of STAT3 in human B cells following IL-21 stimulation. Human B cells were pretreated with vehicle (DMSO) or 10 μM iguratimod for 30 min and then stimulated with IL-21 for 15 min. pSTAT3 (Y705) was detected by flow cytometry. (A) Representative FCM plots and (B) cumulative data of pSTAT3 were shown (n = 3). One representative of at least three independent experiments was shown. Data were shown as mean ± SEM and analyzed by one-way ANOVA (B). ***P &lt; 0.001. Figure S4. Iguratimod inhibits TFs required for ASC differentiation in RNA-seq data. (A) GSEA identified the responses to endoplasmic reticulum stress and unfolded protein response pathways were inhibited by iguratimod in activated human B cells. (B, C) RPKM plots showed the expressions of key TFs in ASC differentiation (B) and maintaining B cell identity (C) from the RNA-seq data derived from vehicle (DMSO) or 10 μM iguratimod-treated activated human B cells (n = 3). Data were expressed as mean ± SEM and analyzed by paired Student’s t test (B, C). *P &lt; 0.05. RPKM, reads per kilobase of exon per million mapped sequence reads. Table S1. Baseline characteristics of the six naive RA patients. Table S2. qRT-PCR primers used in this study (DOCX 9552 kb)