Ma1 710

Mouse myofibroblasts (MFBs) were extracted and cultured from the lungs of 5–7-day-old C57BL6 mice as described previously (27 (link)). Cultured cells were then incubated with TGF-β (5 ng/mL) (100-21, PeproTech) or a combination of TGF-β (5 ng/mL), 10⁻⁶ M all-trans retinoic acid (ATRA; R2625, Sigma-Aldrich), and 10⁻⁷ M 1,25-dihydroxy vitamin D3 (VitD) (D1530, Sigma- Aldrich) for 40 h as previously described (30 (link)). At the end of the experiment, cells were lysed with radioimmunoprecipitation assay buffer (RIPA buffer) and processed for immunoblot analysis (25 (link)). Restore Western Blot stripping buffers (21059, Thermo Fisher) were used to remove the antibodies from the membranes to allow reprobing. Antibodies included PDGFRα (338, Santa Cruz Biotechnology), pERK (4370, Cell Signaling Technologies), pSMAD2-3 (PA5-99378, Thermo Fisher), vitamin D receptor (MA1-710, Thermo Fisher), and retinoid X receptor (5388, Cell Signaling).

Tissues were fixed in 10% buffered formalin for 24 h prior to processing. Tissues were processed and embedded in paraffin and then sectioned at 5 μm. Slides were deparaffinized in several baths of xylene and then rehydrated in graded alcohols followed by ddH₂O. Slides were incubated in 1× pH 6 citrate buffer (Invitrogen, 00-5000) in DAKO PT Link for 20 min. IHC was performed using DAKO Autostainer Plus following manufacturer’s instructions. Slides were incubated in 3% H₂O₂ for 15 min. To block non-specific binding, tissues were incubated with 10% normal goat serum for 30 min, followed by avidin/biotin block (Vector Labs, SP-2001). Primary antibody LSD1 (Cell Signaling, 2139) or VDR (Thermo Scientific MA1710 (Clone 9A7)) were diluted in 1% BSA solution and incubated for 30 min at room temperature, followed by the biotinylated Goat Anti-Rabbit secondary antibody (Abcam, ab6720) for 15 min. For signal enhancement, ABC reagent (Vector Labs, PK-6100) was applied for 30 min. Slides were then incubated with DAB substrate (Dako, K3467) for 5 min and then counterstained with DAKO hematoxylin for 20 s. Slides were dehydrated through several baths of graded alcohols and xylenes and then coverslipped. Finally, slides were scanned using the Aperio System (Leica).