Genetool software

Cells were harvested and lysed in radioimmunoprecipitation (RIPA) buffer as previously described [54 (link)]. Equal amounts of proteins were separated by SDS-PAGE gel electrophoresis (Bio-Rad). Proteins were transferred to an Immobilion-P transfer membrane (Merck Millipore), and detected using standard immunoblotting techniques. Proteins were visualized using SuperSignal™ West Dura Extended Duration Substrate kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Images were captured using a Syngene ChemiGenious camera and presented by the GeneSnap software tool (Syngene, Cambridge, England). Intensity of protein bands was quantified by using the GeneTool software (Syngene).

For immunoblot assay, cells were first subjected to lysis in NuPAGE LDS Sample Buffer. Total proteins content from every samples was measure using Pierce BCA Protein Assay Kit according to the manufacturer’s recommendations. Samples lysates were then loaded onto NuPAGE 4–12% Bis-Tris Protein Gels (10, 12, or 15 wells). After electrophoresis, proteins were transferred to nitrocellulose membranes. The transblotted membranes were blocked for 1 h and then probed with appropriate primary antibodies (dilution as recommended by manufacturers) overnight at 4 °C. Next, the membranes were washed three times for a total of 30 min and then incubated with secondary antibodies at room temperature for 1 hr. After another three washes, proteins were detected using SuperSignal West Pico PLUS chemiluminescent Substrate, PXi imager (Syngene), and GeneSys software (Syngene, Paris, France), according to the manufacturer’s manual. Proteins expression were quantified using GeneTool software (Syngene) and normalized to their corresponding loading control. Antibodies for immunoblotting were purchased from the following sources: FASN (#3180S) from Cell Signaling Technology; Actin (MAB1501) from Millipore; IDH1 R132H (DIA-H09) from Dianova. HRP conjugate anti-rabbit (W4011) and anti-mouse (W4021) secondary antibodies were purchased from Promega (Charbonnières les Bains, France).

Independent samples were used for Western blot including 13 KC (8 males and 5 females) and 8 control (2 male and 6 females) samples. Proteins were extracted by firstly homogenizing the collected epithelial tissues in RIPA buffer (Sigma, USA) supplemented with cOmplete™ Protease Inhibitor Cocktail Tablets (Roche, USA) for 10 min using a homogeniser, followed by centrifugation at 12,000 × g for 15 min and 4 °C. The supernatant was removed, aliquoted and stored at −80 °C until use. Protein concentration was measured using a FluroProfile^® kit (Sigma-Aldrich, USA) as previously published^{8 (link)}. Sixty micrograms of total protein from each sample was used.
Western blotting was performed as previously published^{60 (link)}. Samples were tested for the expression of Notch1, cleaved Notch1 (NICD), plasmolipin (PLLP) and proto-oncogene tyrosine-protein kinase (Src), and GAPDH and Pan-actin were used as the loading control (Table S8). HRP conjugated anti-rabbit IgG secondary antibody (Millipore) was used at 1 in 25,000 dilution. The band volume for each protein (with background subtracted) were analysed using the GeneTool software (Syngene, UK), and the ratio of target proteins/GAPDH was calculated. A two-tailed Student’s t-test was used to determine the significance between KC and control samples (p < 0.05 is considered as significant).