Anti bhlhe40

The clinical tissue samples used in this study were histopathologically and clinically diagnosed at Ruijin Hospital with patient consent and with approval from the Ethics Committee. This information is presented in Table S2 (Supporting Information). IHC staining was performed with the following antibodies: anti‐BHLHE40 (1:100, Novus), anti‐SREBF1 (1:100, Abcam), anti‐SCD1 (1:100, Abcam), and anti‐Ki67 (1:100, Servicebio) to detect protein expression. The protein expressions of BHLHE40, SREBF1, and SCD were analyzed using the digital pathological image analysis software based on artificial intelligence learning (Servicebio Technology) via tracking, color selection, calculation, and TMA. The analysis formula is H‐SCORE = ∑(pi×i) = (percentage of weak intensity ×1) + (percentage of moderate intensity ×2) + (percentage of strong intensity ×3).

For immunoblotting (IB) analysis, whole cell lysates and nuclear proteins were prepared using RIPA buffer supplemented with protease inhibitor cocktails (Sigma-Aldrich) and the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific), respectively. To detect protein-protein interaction, soluble proteins were extracted using the Pierce IP Lysis Buffer (Thermo Scientific) supplemented with protease inhibitor cocktails and co-immunoprecipitation (CoIP) was performed using the TrueBlot Immunoprecipitation and Western Blot Kit (Rockland Immunochemicals Inc., Limerick, PA, USA). IB signals were developed using the SuperSignal West Dura Extended Duration Substrate and the CL-XPosure Film (Thermo Scientific). Antibodies used in this study were: anti-EGFR-Tyr1110P, anti-EGFR, anti-ERK1/2-Thr202/Tyr204P, anti-ERK1/2, anti-AKT-Tyr416P, anti-AKT, anti-Caspase9, anti-HDAC1, and anti-HDAC2 from Cell Signaling Technologies (Boston, MA, USA), anti-GAPDH from Millipore (Merck, Darmstadt, Germany), anti-TBP from Abcam (Cambridge, MA, USA), anti-CTGF from Abgent (San Diego, CA, USA), anti-HBEGF and anti-CD9 from R&D Biosystems (Minneapolis, MN, USA), anti-BHLHE40, anti-HIF1A, and anti-EPAS1 from Novus Biologicals (Littleton, CO, USA), and anti-ALIX, anti-TSG101, and anti-CD81 from Santa Cruz (Dallas, TX, USA).