Rs0002

The primary antibodies purchased from Santa Cruz Biotechnology are specified as follows: anti-STAT6: sc-374021, anti-p-STAT6: sc-136019, anti-Nrf2: sc-13032, anti-NQO1: sc-32793, anti-VDR: sc-13133, anti-CYP24A: sc-365700, anti-Histone H3: sc-517576, anti-Tublin: sc-8035, anti-ATG7: sc-376212, anti-Beclin1: sc-48381, and anti-GAPDH: sc-32233. Antibody against LC3B was purchased from novusbio (NB100-2220). Secondary antibodies conjugated with horseradish peroxidase (HRP) were from Immunoway (Plano, TX: RS0001 (Mouse) and RS0002 (Rabbit)). Human bronchial epithelial cell BEAS-2B and human acute monocytic leukemia cell line (THP-1) were purchased from ATCC (Manassas, VA). BEAS-2B cells were cultured in BEBM with additives from Lonza/Clonetics Corporation (CC-3170), and THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Hyclone, Logan, UT) and 0.1% gentamycin (Invitrogen, Carlsbad, CA). For the differentiation of THP-1 cells, 5 ng/ml phorbol-12-myristate-13-acetate (PMA) obtained from Sigma was used. The cells were maintained at 37°C in a humidified incubator containing 5% CO₂.

Total protein was extracted from the liver tissue and separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred onto polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were blocked with 5% milk for 1 h and incubated overnight at 4 °C with primary antibodies against TLR4 (Proteintech Group, Rosemont, IL, USA; 19811-1-AP), MyD88 (Proteintech Group, 23230-1-AP), NF-κB p65 (Immunoway, Plano, TX, USA; YM3111), phospho-NF-κB p65 (Immunoway, YP0847), and β-actin (Immunoway, YM3028). The membranes were washed three times with phosphate-buffered saline with Tween-20 and incubated with secondary antibodies for an hour. Goat anti-rabbit IgG-HRP (Immunoway, RS0002) and goat anti-mouse IgG-HRP (Immunoway, RS0001) were used as secondary antibodies. Dilutions for primary antibodies and secondary antibodies were 1:1000 and 1:10,000, respectively. Band intensities were quantified using the Image J software (version 1.52) and normalized to β-actin levels. Original blots can be found in the accompanying Source Data file.

A total of 10 paired LUAD primary tumor and para-cancerous tissues of paraffin-embedded samples were provided by the Pathology Department of Xiangya Hospital of Central South University (Hunan, China). All immunohistochemistry (IHC) procedures were performed with the permission of the Ethical Committee of Xiangya Hospital of Central South University. Tissues were fixed in 10% formalin, paraffin-embedded, cut into 3-mm sections, and mounted on slides. After deparaffinization, rehydration, and microwave antigen retrieval, the slides were incubated at 4 °C overnight with an antibody against DPEP2 (Proteintech, 16466-1-AP, 1:100 working dilution, Rosemont, IL, USA). After that, the slides were incubated with secondary antibody (ImmunoWay, RS0002, with 1:1000 working dilution, Plano, TX, USA) at room temperature for 30 min before being stained with DAB (3,3′-diaminobenzidine) substrate (with 1:20 working dilution) and counterstained with hematoxylin. To validate the positive staining IHC results, five random areas in each tissue sample were microscopically examined and analyzed by at least one experienced pathologist. The average staining scores were subsequently calculated by dividing the positive areas by the total areas. The obtained data were expressed as the mean values ± SDs (standard deviation).

Ultimately, we selected the 3-day subgroup to investigate the mechanisms of the fasudil effect in our rat model of CA-AKI. All left kidneys of the three animal groups were collected on Day 3, and the protein concentrations were determined by the bicinchoninic acid method. We then used equal aliquots (∼10 µg) of boiled protein samples for electrophoretic separation (8–12% sodium dodecyl sulfate (SDS) polyacrylamide gel) and western blot. The primary antibodies were: ROCK-1 (1:500, sc-17794; Santa Cruz Biotechnology); ROCK-2 (1:1,000, abs136978; Absin Bioscience); phosphorylated myosin light chain phosphatase (p-MYPT1: 1:1,000, 4,563; Cell Signaling Technology); myosin light chain phosphatase (MYPT1: 1:1,000, abs131546; Absin Bioscience); prolyl-4-hydroxylase domain-containing protein 2 (PHD2: 1:1,000, abs151871; Absin Bioscience); HIF-1α (1:1,000, ab2185; Abcam); TGF-β1 (1:1,000, ab215715; Abcam); Smad3 (1:1,000, 9,523; Cell Signaling Technology); p-Smad3 (1:1,000, 9,520; Cell Signaling Technology); and α-SMA (1:1,000, 19,245; Cell Signaling Technology), followed by peroxidase-labeled conjugated secondary mouse (1:10,000, RS0001; ImmunoWay Biotechnology) or rabbit (1:10,000, RS0002; ImmunoWay Biotechnology) antibodies for 2 h. Specific protein bands were scanned in a western blotting detection device. Band areas were analyzed with ImageJ. The internal control was beta-actin.