Sc 271098

The FISH probes of HSALR1 were purchased from RiboBio Technology Co. Ltd. (China). FISH assay was performed as previously described.¹⁸ The FISH kit (RiboBio) was used to recognize the cellular localization and subcellular localization of HSALR1 following the manufacturer's protocol. Finally, Leica DM6 M was used to visualize the relative fluorescence of cells in each group.
The IF/FISH assay was performed as previously described.⁴⁴ The FISH assay was carried out following the manufacturer's protocol, and subsequently, the lung tissue sections were incubated with antibodies against human FN1 (1:100, sc‐271098, Santa Cruz) and Vimentin (1:100, ab8978, Abcam) for 2 h at 37°C, followed by incubation with Alexa Fluor 488 goat anti‐rabbit IgG (H + L) (1:500, Invitrogen) for 40 min. Images were captured using the Leica DM6 M.

The proteins from glomeruli or cells were fractionated by electrophoresis on 10% SDS-PAGE, electroblotted to PVDF filter membranes, and incubated with the primary antibody at 4 °C and then with a horseradish peroxidase-conjugated secondary antibody. The experiment was repeated three times. Primary antibodies were mouse anti-collagen 1α (ab6308, abcam, USA), rabbit anti-αSMA (ab5694, abcam, Shanghai, China), mouse anti-fibronectin (sc-271098, Santa Cruz, USA), rabbit anti-TGFβ (ab92486, abcam, Shanghai, China), rabbit anti-Smad2/3 (8685 Cell Signaling Technology, Shanghai, China), rabbit anti-proteasome 20S LMP7 (β5i) (ab3329, abcam, Shanghai, China), rabbit anti-pIκB (2895S, Cell Signaling Technology, Shanghai, China), rabbit anti-IκB (4812, Cell Signaling Technology, Shanghai, China), rabbit anti-NFκB (8242, Cell Signaling Technology, Shanghai, China), rabbit anti-pNFκB (3033, Cell Signaling Technology, Shanghai, China), rabbit anti-histone H₃ (9715, Cell Signaling Technology, Shanghai, China), rabbit anti-GAPDH (5174, Cell Signaling Technology, Shanghai, China). The antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin was used to verify equal loading of proteins. Densitometry was performed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Kidneys were embedded in OCT (4583, SAKURATissue-Tek®, Torrance, CA, USA) on dry ice. Five-micrometer sections were cut and performed with immunostaining. Briefly, the slices were fixed in 10% neutral buffered formalin; then washed with PBS and treated with 0.2% Triton X-100; mouse anti-αSMA (MS-113-P, Thermofisher, USA) and rabbit anti-synapotodin (sc-50459; Santa Cruz Biotechnology, Santa Cruz, CA, USA); or mouse anti-collagen 1α (ab6308, abcam, USA) and rabbit anti-synapotodin; or mouse anti-fibronectin (sc-271098, Santa Cruz, USA) and rabbit anti-synapotodin; or mouse anti-TGFβ (MAB1835, R&D, USA) and rabbit anti-synapotodin were incubated with the slices after blocked with 1% BSA; Donkey anti-mouse IgG-488 (ab150105; Abcam, Shanghai, China) and donkey anti-rabbit IgG-647 (ab150075; Abcam) were incubated with the slices; Hoechst 33342 was then used to stain the nucleus; the slices were mounted in VECTASHIELD Mounting Medium (H-1000, Vector Laboratories, Inc., Burlingame, CA, USA) staining. Images were obtained using a confocal microscope (TCS-SP8; Leica, Buffalo Grove, IL, USA). Positive cells with both synapotodin and EMT markers located in the glomeruli were counted and divided by synapotodin-positive cells located in the glomeruli. At least 100 glomeruli were analyzed in each group.