Anti lsd1

Western blot analysis were carried out according to the standard procedures [8 (link)] for anti-TNC (1:1000, Abcam, UK), anti-LSD1 (1:2000, Sigma, USA), anti-SOX2 (1:1000, Abcam, UK), anti-CD44 (1:1000, Millipore, USA), anti-CD133 (1:1000, NOVUS, USA), anti-SOX9 (1:1000, Abnova, USA), anti-GLI1 (1:1000, Abcam, UK), and anti-β-actin (1:500, Bioss, China).

After separation in SDS-PAGE (AA: 10%, AA/BisAA ratio: 36:1), the proteins were transferred onto PVDF membranes following overnight incubation with specific primary antibodies. The following primary antibodies were used: anti-actin (Sigma A5441, Sigma-Aldrich, St. Louis, MO, USA), anti-Zeb1 (Sigma AMAb90510, Sigma-Aldrich, St. Louis, MO, USA), anti-Nanog (Santa Cruz sc-293121, Dallas, TX, USA), anti-Oct4 (Cell Signaling #2890s, Danvers, MA, USA), anti-Sox2 (Cell Signaling #3579s, Danvers, MA, USA), anti-CTBP2 (Abcam ab128871, Cambridge, UK), anti-CTBP1 (Sigma HPA018987, Sigma-Aldrich, St. Louis, MO, USA), anti-LSD1 (Sigma ABE365, Sigma-Aldrich, St. Louis, MO, USA), anti-TRIM33 (Sigma HPA004345, Sigma-Aldrich, St. Louis, MO, USA), and anti-p53 (Cell Signaling #46565, Danvers, MA, USA). The secondary antibodies were from Sigma: anti-mouse (A9917) and anti-rabbit (A0545). Bound antibodies were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate ECL kit (Thermo scientific, Waltham, MA, USA), chemiluminescence was detected using ChemiDoc (BioRad, Hercules, CA, USA).

Western blotting was performed as previously described [57 (link)]. Briefly, 60 μg protein was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% skim milk for 2 hours and incubated for 15 hours with the following rabbit monoclonal primary antibodies: anti-LSD1 (diluted 1:500; Sigma, St. Louis, MO, USA), anti-HPV16 E7 (diluted 1:100; Bioss, Shanghai, China), anti-Vimentin (diluted 1:500; Cell Signaling Technology, Beverley, MA, USA), anti-E-cadherin (diluted 1:500; Cell Signaling Technology), anti-GAPDH (Epitomics), anti-H3K4me1 (Abcam, Cambridge, UK), anti-H3K4me2 (Abcam) and anti-H3K9me2 (Abcam), followed by 1 hour of incubation with the appropriate secondary antibody.

IHC staining and evaluation of the IHC analysis on CRC tissue microarray samples was performed as previously described [8 (link)]. The primary antibody include anti-TNC (1:100, Abcam, UK), anti-SOX2 (1:100, Abcam, UK), anti-CD44 (1:80, Millipore, USA), anti-CD133 (1:100, NOVUS, USA), anti-LSD1 (1:250, Sigma, USA), anti-SOX9 (1:100, Abnova, USA), anti-p21 (1:100, Millipore, USA), anti-cyclinD1 (1:100, Millipore, USA), anti-p27 (1:100, Millipore, USA), anti-CDK4 (1:100, Millipore, USA), anti-p16 (1:100, Millipore, USA), anti-SMO (1:250, Santa, USA), and anti-GLI1 (1:100, Abcam, UK).

After routinely dewaxing and hydration, sections proceed to be antigen repaired with TE buffer at 98 °C. Each section was blocked with 3% H₂O₂. Each section was incubated with anti-Gli1 (Abcam), anti-CD44 (Abcam), anti-LSD1 (Sigma), anti-Sox2 (R&D), anti-Sox9 (Abnova), anti-LGR5 (Abcam), in primary antibody dilution buffer for 1 h at ambient temperature (AT). Then anti-mouse/rabbit antibody were used to incubated with tissue samples for 30 min at AT. Lastly, chromogenic agent 3, 3′-diaminobenzidine (Dako) was used to stain tissue samples.
The double immunostaining procedure was executed in the same section, the first step was to use anti-Gli1 antibody staining with 3,3′-diaminobenzidine, the second step was to use anti-CD105 antibody (Abcam) staining with AEC.
Two pathologists (WB Qi & YH Xuan) assessed the immunohistochemical results and the staining results were assessed according to previous study [9 (link)].