Ripa lysis buffer

Human normal epidermal keratinocytes from donors of various ages (for details, see Supplementary Table S3) (Biopredic International, Rennes, France) were cultured in Humedia-KG2 (KURABO, Osaka, Japan). Human dermal fibroblasts from donors of various ages (for details, see Supplementary Table S4) (Biopredic International, Rennes, France) were cultured in DMEM containing 10% FBS (Gibco, Tokyo, Japan). Culture flasks were incubated at 37 °C in a humidified atmosphere with 5% CO₂. 2.5 × 10⁵ keratinocytes or fibroblasts were seeded into 6-well plates coated or not coated with iMatrix-511, and incubated for 24 h. RNA was extracted from each sample for quantitative PCR analysis and protein was extracted using RIPA lysis buffer (Nacalai tesque, Kyoto, Japan) for protein array analysis.

Syncytin‐2 expression plasmids were transfected into 293T and G355‐5 cells in the same manner as cell fusion assays. Cells were lysed in RIPA Lysis Buffer (#08714‐04; Nacalai Tesque) from transfection to G355‐5 cells at 7 h and from transfection to 293T cells at 20 h. Cellular suspensions were subjected to glycolysis treatment for 1 h using a PNGase F Kit (New England BioLabs). SDS/PAGE was performed using Mini‐PROTEAN TGX Precast Gels (#4561094; Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Peptides from the gel were transferred to polyvinylidene difluoride membranes, and the monoclonal ANTI‐FLAG M2 antibody (#F3165; Sigma‐Aldrich) was used to detect Flag‐tagged syncytin‐2. Signals were detected using a Super Signal West Femto System (#34095; Thermo Fisher Scientific), and images were obtained using a LAS4000 Mini camera system (Fujifilm, Tokyo, Japan).

Protein lysates were extracted from cultured cells 72 h post-treatment using RIPA lysis buffer (Nacalai Tesque, USA) according to the manufacturer's instructions. The protein samples were quantified using the NanoDrop ND-1000 spectrophotometer (ThermoScientific, USA) at 280 nm wavelengthand loaded at 40 mg/well into10-15%bisacrylamide gel (Nascalai Tesque, Japan). Protein samples were then transferred from the gel onto the Immobilon-polyvinylidene fluoride transfer membrane (Millipore, Watford). Blocking solution (5% milk powder and 0.1% Tween-20 in PBS) was used to immerse the membrane for 1 h at room temperature. Then, the membrane was probed with primary antibodies (Notch 1 ICD, Vimentin, E-Cadherin, and Activated caspase-3 (Santa Cruz, USA); Cleaved PARP (Cell Signalling Technology, USA); b-Actin (Sigma Aldrich, USA)) overnight at 4 °C. The membrane was washed in PBST three times (10 min each) and probed with appropriate HRP-conjugated secondary antibodies. The protein bands were detected using Chemi-Lumi one super detection reagents (Nacalai Tesque, USA) and visualised using the C-Digit blot scanner (Lincoln, Nebraska, USA). Image J was used to measure the relative density of each peak with the size and intensity of each band on the blot and normalised to the loading control (b-Actin). Analyses were performed on data from three independent experiments.

To confirm the precise insertion of constructs and expression of HSF1 proteins, genomic PCR and western blotting were performed. The genomic DNA of Pv11 cells and the clonal cell lines was extracted, then subjected to PCR. The primer sequences used in genomic PCRs are shown in Data S1 and S2. Western blotting was performed as described previously [33 (link)]. Briefly, cells were lysed with RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) for 30 min at 4 °C. After centrifugation, aliquots of the supernatant were subjected to protein quantification with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and 20 μg protein/lane was used for SDS-PAGE. After transferring to a PVDF membrane, the membrane was blocked with 1% skimmed milk in TBS with 0.1% Tween 20 (TBST) at 4 °C overnight. Anti-PvHSF1 antibody was generated against peptide ETMNRVLHEVKNMRGRQ in a rabbit (Merck) and used 1:2000 in 1% skimmed milk at room temperature for 1 h. After washing the membrane with TBST, secondary antibody (goat anti-rabbit IgG (H+L) HPR 65-6120, Thermo Fisher Scientific) was used 1:2000 in 1% skimmed milk at room temperature for 1 h. After washing the membrane, chemiluminescent signals from ECL Prime detection reagents (Cytiva, Little Chalfont, Buckinghamshire, UK) were captured on a ChemiDoc™ Touch imaging system (Bio-Rad, Hercules, CA, USA).

Immunoblotting was performed as previously described in detail [17 (link)]. Briefly, cells were lysed with RIPA lysis buffer (Nacalai Tesque) containing 1 mM PMSF, 0.15 U/ml aprotinin, 10 mM EDTA, 10 ng/ml sodium fluoride and 2 mM sodium orthovanadate. Cellular proteins were quantified using a DC Protein Assay (Bio-Rad, Richmond, CA). Equal amounts of proteins were loaded onto the gels, separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). The membranes were probed with primary antibodies (Abs) such as anti-microtubule- associated protein 1 light chain 3 (LC3) B antibody (Ab) (NB600-1384; Novus Biologicals, Inc., Littleton, CO), and anti-phosoho-eIF2α (Ser51) Ab (#9721S), anti-CHOP (GADD153) monoclonal (m) Ab (#2895S), anti-p70S6K Ab (#9202S), anti-phospho-p70S6K (Thr389) Ab (#9205S), anti-PARP Ab (#9542) (Cell Signaling Technology, Danvers, MA, USA), and anti-p62 (sequestosome-1) mAb (sc-28359), anti-GAPDH mAb (sc-32233), and anti-β-actin mAb (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-XBP-1s Ab (S647501) (BioLegend, San Diego, CA). Immunoreactive proteins were detected with horseradish peroxidase-conjugated second Abs (Jackson, West Grove, PA) and an enhanced chemiluminescence reagent (ECL) (Millipore). Densitometry was performed using a Molecular Imager, ChemiDoc XRS system (Bio-Rad).