Pd98059

The renal fibroblasts were stimulated with UTI89, MG1655, CFT073 wild-type and mutants at a multiplicity of infection (MOI) of 10, Adenosine triphosphate (100 µM, ATP; Sigma-Aldrich), Adenosine (100 µM, Sigma-Aldrich), lipopolysaccharide (1 µg/ml, ultrapure LPS-B5, Invivogen, San Diego, CA, USA), flagellin (1 µg/ml, Invivogen) and Pam3CSK4 (1 µg/ml, Enzo Life Sciences, Lausen, Switzerland) for 6 hours and incubated at 37 °C with 5% CO₂. The fibroblasts were also pre-incubated with DMSO (vehicle), p38 MAPK inhibitor SB203580 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), ERK1/2 inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc), NF-κB inhibitor BAY 11-7082 (5 µM, Enzo Life Sciences) and the PKC inhibitor bisindolylmaleimide I (10 µM, Santa Cruz Biotechnology Inc) for 1 hours prior to CFT073 stimulation for 6 hours at MOI 10. Supernatants and mRNA were collected and kept at −80 °C until further analysis.

The 4th through the 10th HFDPC population doublings were seeded at 3 × 10⁴ cells/well in triplicate in 24-well plates. HFDPC were incubated with serum-free basal medium for 18 h and then cultured in medium containing 0.5% fetal calf serum with various concentrations of ebastine in the presence or absence of 5 μM AKT1/2 kinase inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 50 μM PD98059 (Santa Cruz Biotechnology) for an additional 24 h. Cells were cultured in serum-free basal medium or incubated with complete growth medium as negative and positive controls, respectively. A WST-1 assay was used to analyze cell viability. Briefly, 100 μL of WST-1 reagent (Roche Diagnostics, Mannheim, Germany) was added to each well, and the HFDPC were further incubated at 37°C for 4 h. Cells treated with WST-1 were then transferred to a 96-well plate, and the absorbance of each well was measured at wavelengths of 450 and 650 nm in a multifunctional microplate reader (Infinite F200, Tecan, Durham, NC, USA). The percentage of cell viability relative to the vehicle-treated cells was calculated as follows: [(A450–A650) of the drug-treated cells/(A450–A650) of the vehicle-treated cells] × 100%.

Immortalized tubular epithelial cells (HK-2) were cultured in DMEM/F12 medium supplemented with 10% FBS. After synchronization, cells were treated with 20% FSGS patients’ serum (PS) or 40 nM C3a (204881, Merck-Calbiochem). For intervention studies, 1 μM of C3aR antagonist SB290157 (sc-222291, Santa Cruz), 100 μg/ml of eculizumab (Soliris, Alexion Pharmaceuticals), 10 μM of p38 MAPK inhibitor SB203580 (sc-3533, Santa Cruz), 50 μM of ERK inhibitor PD98059 (sc-3532, Santa Cruz), 10 μM of Akt inhibitor MK2206 (sc-364537, Santa Cruz) or 1 μM of U-46619 (sc-201242, Santa Cruz) was given 30 min before treatments. To infect HK-2 cells with plenti-CMV-LOC105375913, plenti-CMV-snail or plenti-CMV-XBP-1s plasmid, the lentiviral stock was mixed with polybrene (1 µg/ml) and added to cells. C/EBPβ siRNA (sc-44251), Elk-1 siRNA (sc-35290), ERα siRNA (sc-29305), GR siRNA (sc-35505), snail siRNA(sc-38398) and XBP-1s siRNA (sc-38627) were bought from Santa Cruz (Dallas, Texas, USA). LOC105375913 siRNA was bought from Thermo fisher (4390771, Carlsbad, California, USA). Transfection of siRNA, miRNA mimics or miRNA antisense oligonucleotide (ASO) was conducted with Lipofectamine 2000.

NF-κB inhibitor Caffeic acid phenethyl ester (CAPE) was purchased from Sigma. ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580 were purchased from Santa Cruz.