Fig. 4: CAV1 regulates distinct molecular pathways in primary and acquired TKI resistance.

A Western blot for key proteins (CAV1, EGFR, FGFR4, E-cadherin, p21 & p27) and pathways (AKT/ERK) in Huh-7 parental and Huh-7/LR cells under basal conditions and post-treatment (n = 3). B Pathway identification following CAV1 knockdown in Huh-7/SR1 cells using a proteome profiler array, quantified by band densitometry (n = 1). C Validation of P70S6K signalling in CAV1-depleted Huh-7/SR1 cells by Western blot (n = 3). D Western blot evaluation of pathways activated by CAV1 overexpression in TKI-naïve Huh-7 parental cells (Huh-7-CAV1-OE) and primary TKI-resistant PLC-PRF-5 cells (PLC-PRF-5-CAV1-OE) (n = 3). E RT-qPCR analysis of CAV1, p21(CDKN1A), CDH1 (E-cadherin) mRNA, with immunofluorescence detection of FGFR4 and E-cadherin in Huh-7, Huh-7/SR1 and Huh-7/LR cells (n = 3). F AXL profiling in Huh-7/SR cells by RT-qPCR and Western blot (n = 3). G Western blot validation of CAV1 dependence in CAV1-depleted Huh-7/SR1 cells treated with sorafenib or DMSO control, further confirmed by STAT3 and NFκB activity assays using luciferase reporters. H Western blot validation of CAV1 dependence in CAV1-depleted Huh-7/LR cells treated with lenvatinib or DMSO control. I Dose-response curve of AXL inhibitor; BGB324, in Huh-7/SR1 cells, assessed by cell viability assay, with synergy between sorafenib and BGB324 evaluated through combination index (CI) and isobologram analysis (Fa = 0.811, CI = 0.68723) and confirmed via IncuCyte growth curve assays (n = 3). J Dose-response curve of selective FGFR4 inhibitor; BLLU9931, in Huh-7/LR cells, with synergy between lenvatinib and BLU9931 assessed via CI and isobologram analysis (Fa = 0.97699, CI = 0.88594), and validated using IncuCyte growth curve assays (n = 3). K Western blot analysis for autophagy markers (P62, LC3A/B I (16 kDa), LC3A/B II (14 kDa), and LAMP1) in CAV1-depleted Huh-7/SR1 cells following sorafenib treatment. All experiments were performed on three independent days with at least three technical replicates. Error bars represent ± SD. Growth curves and time course studies were analysed by one-way repeated measure ANOVA, while all other data were evaluated by one-way ANOVA with multiple comparison ( > 2 groups) or an unpaired two-tailed student’s t test. Significance is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CI = combination index, Fa = fractional inhibition, SF = sorafenib, Lenva = lenvatinib. β-actin and α-tubulin were used as loading controls.