Bca1 1kt

Western blot analysis was performed to detect the protein expression levels of Bax, Bcl-2 and HSPA5. Proteins were extracted from the DLBCL cells treated with 17-DMAG and from untreated control cells. The cells were plated in 6-well culture dishes at a density of 6×10⁵ cells/well. The harvested cells were then lysed on ice for 30 min in 100 ml of lysis buffer [120 mmol/l NaCl, 40 mmol/l Tris (pH 8), 0.1% NP40] and centrifuged at 8,600 × g for 30 min at 37°C. The bicinchoninic acid assay method (BCA1-1KT, Sigma Aldrich; Merck KGaA) was used to determine the protein concentration of the samples. Protein samples (20 µg/lane) were resolved using 10% SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were subsequently incubated with the appropriate primary antibodies against Bax (1:1,000), Bcl-2 (1:1,000) and HSPA5 (1: 1,000) to detect each the proteins of interest separately at 4°C overnight. β-actin (1:1,000) was used as a loading control. After being washed with PBS, the membranes were incubated with peroxidase-conjugated secondary antibodies (ab6728, Abcam, Cambridge, UK) for 2 h at room temperature. Finally, the detected protein bands were visualized using an Immobilon Western Chemiluminescent HRP Substrate kit (Merck KGaA) and the ImageQuant LAS 4000 software (GE Healthcare Life Sciences, Little Chalfont, UK).

HepG2 cells were seeded at a density of 5 × 10⁴ cells/well in six wells of the eight-well Seahorse plate and grown for 48 h. For the OCR assay, the medium was replaced with Seahorse XF DMEM Medium, pH 7.4, supplemented with 10 mM d-Glucose, 1 mM pyruvate and 2 mM l-Glutamine, and cell cultures were allowed to equilibrate for 1 h at 37 °C in a no-CO₂ incubator. For the ECAR assay, the medium was replaced with Seahorse XF DMEM Medium, pH 7.4, supplemented with 4 mM l-Glutamine. Seahorse assays were performed according to the manufacturer's instructions, as reported in Pasquale et al. [33 (link)]. At the end of the assay, protein from each well was obtained for both the OCR and ECAR assays was extracted to normalise for variation in cell number. Briefly, cells were lysed with 1X lysis buffer (Cell Signalling Technology, #: 9803S), and lysates were centrifuged at 17,000 g at 4 °C for 10 min. Protein concentrations were then quantified using the bicinchoninic protein assay (Sigma #: BCA1-1 KT).

Total protein was extracted from cells using 200 µL 1 × SDS lysis buffer (P0013G; Beyotime Biotechnology Co., Shanghai, China) containing protease inhibitor. The concentration was determined using a BCA protein assay kit (BCA1-1KT; Sigma). Next, 30 µg protein was separated with 5% SDS-PAGE and transferred onto membranes. The membrane was blocked with 5% skimmed milk-TBST for 1 hour and probed overnight at 4°C with primary antibodies to HNF4A (ab92378; Abcam Inc., Cambridge, UK), CACNA1A (ab181371; Abcam), VEGFA (ab46154; Abcam), and β-actin (mAbcam 8226, 1: 5000; Abbkine, Redlands, CA, USA). After washing, the membrane was re-probed with the HRP-conjugated secondary antibody goat anti-rabbit (A0208; Beyotime) at 37°C for 45 minutes. Afterward, the membrane was visualized using an ECL reagent (ECL808-25; Biomiga), and the band intensities were analyzed using Gel-Pro Analyzer version 4.0 software (Media Cybernetics, Silver Springs, MD, USA). The ratio of the gray value of the target band to the internal reference β-actin was representative of the relative protein expression.