Ab53278

Formaldehyde (4%) fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1h at 56 °C and immunohistochemical staining performed with the automated instrument Discovery XT (Ventana medical system, Tucson Arizona, USA) using the Chromomap DAB Detection kit as follow: sections were deparaffinized and rehydrated with EZ prep (Ventana) and washed with Reaction buffer (Venatana). The antigens were retrieved with heat treatment in pH 6.0 Citrate buffer (Ribo CC, ventana) at 90 °C for 30 min for anti-Ki-67 (ab15580; Abcam, Cambridge, United Kingdom), CK-19 (ab52625, Abcam, Cambridge, UK), α-SMA (ab5694, Abcam), 4-HNE (ab46545, Abcam) and ALDH7A1 (ab53278, Abcam).

For immunofluorescence staining, cells were seeded on coverslips and after 24 h, the cells were treated as indicated. After 48 h, cells were fixed with 4% (w/v) paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 10 min. After blocking with 3% BSA in PBS, the cells were stained with anti-4-hydroxynonenal polyclonal antibody (ab48506; Abcam, Cambridge, United Kingdom) or ALDH7A1 antibody (ab53278; Abcam, Cambridge, United Kingdom) overnight at 4 °C and then in Alexa Fluor 594-conjugated anti-mouse antibody (A11032; Life Technologies, Carlsbad, CA, USA) or Alexa Fluor 488-conjugated anti-rabbit antibody (A21206; Life Technologies, Carlsbad, CA, USA) diluted in 3% BSA in PBS for 1h at room temperature in dark. The cells were then stained with Hoechst 33342 (H1399, Thermo Fisher Scientific, Waltham, MA, USA) 10 min in PBS to visualize nuclei. The samples were examined under a Zeiss LSM780 confocal microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany).

Harvested cells were lysed with RIPA cell lysis buffer in the presence of protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA). The protein concentration of the cell lysates was quantified by a BCA Pierce Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The same amount of protein samples was loaded onto 10% SDS-PAGE and transferred onto PVDF membranes. After blocking by 5% BSA, the membranes were incubated in the primary antibodies diluted in 5% BSA buffer for overnight at 4 °C and then in the HRP-conjugated secondary antibody for 1 h at room temperature. The protein band images were captured with ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies used in the experiments were ALDH1L1 (ab175198, Abcam, Cambridge, UK), ALDH1A3 (ab129815, Abcam), ALDH1L2 (ab113496, Abcam), ALDH2 (ab108306, Abcam), ALDH3A1 (ab76976, Abcam), ALDH3B1 (SAB4500866, Sigma-Aldrich), ALDH4A1 (ab185208, Abcam), ALDH7A1 (ab53278, Abcam), Flag (F1804, Sigma-Aldrich) and β-actin (sc-47778, Santa Cruz Biotechnology).