Cell line: Human Adipose tissue-derived MSCs (hAD-MSCs) was commercially purchased from Lonza company (Cat# PT5006, Lot# 21TL138912). MSCs were expanded using Dulbecco’s Modified Eagle’s Medium Low Glucose (DMEM-Low Glucose, Euroclone) which contained 5.6 mmol/L glucose and were supplemented with 10% fetal bovine serum (FBS, Gibco) [15], 0.1 mg/ml streptomycin, and 100 units/ml penicillin in standard cell culture incubator (5% CO2/95% air; 37 °C). Medium was changed every 72 h and cells were passaged when confluency was over 70%.

Hypoxia induction: Cobalt Chloride (CoCl2) was used as a chemical inducer of hypoxia [16]. CoCl2 solution was added in the MSCs culture media at a final concentration of 400 μm. MSCs were incubated in the culture media having CoCl2 for 48 h. MSCs grown in normoxic condition (5% CO2/95% air; 37 °C) were considered the control group.

Antibodies used for Western blotting and Immunostaining: HIF-1α (Santa Cruz Biotechnology, US, Cat# sc-53546), TNF-α (Santa Cruz Biotechnology, Cat# sc-52746), PI3K (Santa Cruz Biotechnology, Cat # sc-1637), Caspase-3 (Santa Cruz Biotechnology, Cat # sc-56053), Factor H (MyBiosource Cat # MBS8565537), CD59 (MyBiosource Cat # MBS210610), C5b (MyBioSource, Cat # MBS530209), C9 (R & D system Cat # MAB8126), β-actin (Santa Cruz Biotechnology Cat # sc-47778 HRP), Phalloidin-iFluor 488 Reagent (Abcam, Cat # ab176753), Anti-mouse Alexa Fluor 647 (Invitrogen, USA), Anti-rabbit Alexa Fluor 647 (Invitrogen, USA), Mounting Medium With DAPI (Abeam, UK), Ultra High Sensitivity ECL Kit (GlpBio Cat# GK10008).

The protein levels of PI3K, Caspase-3, Factor H, CD59, C5b, and C9 were measured by Western blotting. Briefly, hAD-MSCs were seeded in 10 cm dishes and cultured for 96 h until getting confluent, then they exposed to hypoxia as described above. The cells were scraped using cold phosphate buffered saline (PBS) and pelleted. The cells pellet was re-suspended in protein lysis buffer (RIPA with protease inhibitors). Total protein levels were measured using NanoDrop™ Lite Spectrophotometer, and 40 μg of protein was loaded onto SDS–PAGE. Following electrophoresis, proteins were transferred to PVDF membrane and were incubated with appropriate primary and secondary antibodies. The membranes were visualized using VILBER FUSION Gel Documentation System, and bands were quantified using ImageJ for densitometry and normalized to β-actin.

hAD-MSCs cells were seeded onto sterile coverslips and allowed to grow till 60% confluency. After being exposed to hypoxic stress for 48 h, the plated cells were fixed with 4% paraformaldehyde and permeabilized using 0.2% Triton X in PBS at RT. The cells were then stained with respective primary and secondary fluorescent antibodies, Thereafter, the stained cells were preserved and counter-stained with Mounting Media having DAPI (4′,6-diamidino-2-phenylindole) for nuclei. The cells were imaged using Cytation 5 imaging system (BioTek, USA).

ELISA assays were performed to detect the expression and activity of C3b (Genochem world SL., lot # GW3605Hu), iC3b (My BioSource, Cat # MBS1603937), and MAC complex (C5b-C9) (GenoChem, Cat # GW3858Hu), following the manufacturer's instructions. Briefly, MSCs were seeded in 96 well plate (1X 105 cell/well) and allowed to attach overnight. The attached cells then exposed to hypoxic stress for 48 h, followed by performing the ELISA assays. The absorbance values which represent the level of each complement factor were detected using Cytation 5 (BioTek, USA).

Data were reported as mean ± SD. Comparison of data between multiple groups was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test, and analysis between two groups was made using Student’s t-test (two-tailed). Statistical significance is determined as p < 0.05. Each figure represents one of at least three independent quantifiable experiments.