Additional file 1 of Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α
Creators: name: Gong, Biao
name: Guo, Duancheng
name: Zheng, Chaonan
name: Ma, Zhen
name: Zhang, Jie
name: Qu, Yanghui
name: Li, Xinhua
name: Li, Gen
name: Zhang, Li
name: Wang, Yuan
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Fig. S1. C3a is present in the tumor tissue of MB patients and C3aR is present on both astrocytes and microglia. a, IHC staining of C3a on human MB sections derived from 4 patients, respectively (P#2–P#5). b, Representative immunostaining of C3aR (in red) and GFAP (in green) or Iba-1(in green) on human MB sections. Fig. S2. C3aR antagonist SB290157 inhibits GFAP expression by C3a-administrated astrocytes. Primary astrocytes were stimulated with C3a (100 nM) with or without SB290157 (2 μΜ) for 48 h in vitro. Immunostaining of GFAP (green) was performed (a), and the percentage of GFAP + cells was quantified (b). Fig. S3. C3a does not activate the Erk pathway in astrocytes. Primary astrocytes were stimulated with 100 nM C3a for 2, 5, 10 and 20 min. Then, the cells were harvested and lysed for total Erk and phosphorylated Erk (p-Erk) detection by western blotting. β-Tubulin served as a protein sample loading control. Fig. S4. Phosphorylation of p38 in C3a-administrated astrocytes is inhibited by C3aR antagonist SB290157. a, Primary astrocytes were stimulated with C3a (100 nM) with or without addition of SB290157 (2 μΜ) for 4 h in vitro. Then, the cells were harvested and lysed for total p38 and phosphorylated p38 (p–p38) detection by western blotting. Fig. S5. GFAP and TNF-α expression and the phosphorylation of p38 in C3a-administrated astrocytes is inhibited by C3aR antagonist SB290157. Primary astrocytes were stimulated with C3a (100 nM, from R&amp;D) in the presence or absence of SB290157 (2 μΜ) for 48 h (a and b), 4 h (c) and 12 h (d), respectively, in vitro. a, Immunostaining of GFAP (in green) was performed, and b, the percentage of GFAP + cells was quantified. c, the cells were harvested and lysed for total p38 and phosphorylated p38 (p–p38) detection by western blotting. d, the cells were harvested to perform qPCR for evaluating TNF-α mRNA levels. ***p &lt; 0.001 vs. C3a (−) &amp; SB290157 (−) group, ##p &lt; 0.01 and ###p &lt; 0.001 vs. C3a ( +) &amp; SB2901570 (−) group. Fig. S6. C3aR antagonist treatment inhibits the p38 MAPK pathway activation in tumor tissue of subcutaneous MB mice. Tumor tissue of C3aR antagonist (SB290157)- and vehicle (MCT)-treated subcutaneous MB mice was harvested at the termination of treatment, and tumor tissue was homogenized to evaluate phosphorylated p38 (p–p38) and total p38 levels by western blotting. β-Tubulin served as a protein sample loading control. Fig. S7. TNF-α receptor is expressed on MB cells. Primary MB cells (pMB), TAAs and BV2 cells were harvested and lysed for RT–PCR assays to detect TNF-α receptor (TNFSF1A) expression. GAPDH served as an internal control.