Fig 2: Prohibitin is reduced in GBM cells following CBD treatment. A) After 1 h treatment with CBD (5 μM), PHB protein levels were reduced in LN18 cells compared to DMSO treated controls; a = 11.3%, b = 37.7% reduction of PHB protein in CBD treated versus DMSO control treated cells. B) In LN229 GBM cells, reduced PHB protein levels were also observed after 1 h CBD treatment, compared to DMSO treated control cells; a = 15.7% and b = 15% reduction of PHB protein after CBD treatment respectively. C) Reduced PHB protein levels (3–8%) were also observed in LN18 cells treated for 1 h with CBD (5 μM) in combination with TMZ (800 μM), compared to TMZ treatment alone. D) In LN229 cells, reduced PHB protein levels (5–9%) were also observed in CBD-TMZ versus TMZ treatment alone. “R” indicates the change in PHB protein levels relative to β-actin, which was used as the internal loading control.

Fig 3: Src-mediated phosphorylation disrupts PHB1/2 complex stability and triggers PHB2 cytoplasmic mislocalization. A Electrostatic surface potential analysis of PHB2 showing the effect of phosphorylation. Left panel: wild-type PHB2 with Y34 and Y77 residues. Right panel: phosphorylated PHB2 (phos-Y34 and phos-Y77) showing altered electrostatic distribution. Blue indicates positive charge (+5), white is neutral (0), and red indicates negative charge (−5). The double-framed line represents the inner mitochondria membrane. B Effect of Src-mediated phosphorylation on PHB1/2 complex formation. Blue Native PAGE (BN-PAGE) analysis of PHB2-WT and phosphorylation-resistant mutant PHB2–Y34F/Y77FFlag in HepG2 cells with or without SRC overexpression. Protein load was verified by Coomassie blue (CBB) staining. Lower panel shows SDS-PAGE verification of SRC, PHB1, PHB2, and β-Actin expression. C Analysis of PHB1/2 complex formation in LO2 versus HepG2 cell lines. Upper panel: representative BN-PAGE showing PHB1/2-SC (supercomplex) and PHB1/2 complex formation detected with PHB2 and AFG3L2 antibodies. Lower panel: SDS-PAGE Western blot analysis of PHB2, AFG3L2, and β-Actin (loading control) expression levels. D Effect of OXPHOS inhibitors on PHB1/2 complex formation. Left panels: representative BN-PAGE analysis of PHB1/2-SC and PHB1/2 complexes in control and treated conditions (H2O2, Rotenone, Antimycin, Oligomycin (OLG), and combined Antimycin/Oligomycin (OA)). Protein load was verified by Coomassie blue (CBB) staining. Lower panel: representative Western blot showing protein expression levels of PHB2, AFG3L2, TIM23, and β-Actin (loading control). E Subcellular distribution of PHB2 under tumor microenvironment stresses. Western blot analysis of PHB2 in mitochondrial and cytoplasmic fractions of HepG2 cells treated with H2O2 (oxidative stress), CoCl2 (hypoxia mimetic), rotenone (Complex I inhibitor), or antimycin (Complex III inhibitor). F Dose-dependent PHB2 redistribution in response to increasing SRC expression levels. Western blot analysis showing PHB2 subcellular localization in mitochondrial and cytoplasmic fractions after SRC overexpression in HepG2 cells. HSP60 serves as mitochondrial marker and β-Actin as cytoplasmic marker (E, F). All Blue Native PAGE and Western blot experiments (B–F) were performed in at least three independent experiments. Representative images are shown.

Fig 4: PHB2 N-terminal residues mediate cardiolipin enrichment on the inner leaflet to regulate the OMA1-OPA1 proteolytic axis and cristae structure. A,B Radial distribution function analysis of phospholipid interactions on the IMM-inner leaflet. (A) PHB1 N-terminal residues (Lys4) and (B) PHB2 N-terminal residues (Gln3, Lys6, Gly10, Arg11, Gly15, Arg17) showing preferential interaction with cardiolipin versus PC/PE. Peak interactions occur at ∼0.5 nm distance. C,D Temporal evolution of phospholipid density distribution on the IMM-inner leaflet. (C) Initial state (0-0.1 μs) and (D) final state (3.9-4.0 μs) showing cardiolipin enrichment (upper panels) versus PC/PE distribution (lower panels) around the PHB1/2 ring periphery. E,F Interaction dynamics between cardiolipin and N-terminal residues. (E) PHB1 subunit interactions with Lys4. (F) PHB2 subunit interactions with multiple residues (Gln3, Gly10, Gly15, Lys6, Arg11, Arg17). Color gradients indicate interaction frequency over 4 μs simulation. G Effect of PHB2–K6A/R17A mutations on PHB1/2 complex formation. HEK293T-PHB2−/− cells expressing Flag-tagged PHB2-WT or PHB2–K6A/R17A were subjected to anti-Flag immunoprecipitation and elution, followed by Blue Native PAGE analysis to detect PHB1/2 and PHB1/2-SC. In: Input; E: Elution. Protein load was verified by Coomassie blue (CBB) staining. H,I OMA1 and OPA1 processing analysis. (H) Western blot showing OMA1 isoforms (pre-pro-OMA1, L-OMA1, S-OMA1) in cells expressing PHB2-WT or PHB2–K6A/R17A. Quantification shown in bar graph (n = 3). (I) OPA1 cleavage analysis showing L-OPA1 and S-OPA1 ratios with quantification (n = 3). J TEM analysis of mitochondrial cristae structure. Representative images and quantification of mitochondrial area and cristae density in HEK293T cells expressing PHB2-WT or PHB2–K6A/R17A, combined with control (NC-shRNA) or OMA1 knockdown (OMA1-shRNA). n = 100 mitochondria per group, pooled from three independent experiments. Scale bars: 1 μm. All statistical data represent mean ± SD, analyzed using unpaired Student's t-test (H, I), and two-way ANOVA (J). ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

Fig 5: Src kinase specifically phosphorylates PHB2 at Y34 and Y77 in hepatocellular carcinoma cells. A,B Differential phosphorylation of PHB proteins in LO2 versus HepG2 cell lines. (A) PHB1 phosphorylation analysis. (B) PHB2 phosphorylation analysis. Upper panels: Immunoprecipitation (IP) with anti-PHB1 or anti-PHB2 antibodies followed by detection of phosphorylated proteins (phos-PHB1/2) and total PHB1/2. IgG serves as negative control. Lower panels: Input lysates showing total PHB1/2 and β-actin loading control. C Identification of kinases involved in PHB2 phosphorylation in HepG2 cells. HepG2 cells expressing PHB2-WTFlag were transfected with siRNA targeting INSR, SRC, or EGFR, or scramble control. Upper panel: IP with anti-PHB2 antibody followed by Western blot detection of phos-PHB2, total PHB2, and IgG control. Lower panel: Input lysates showing PHB2 and β-Actin (loading control). D Effect of SRC overexpression on PHB2 phosphorylation in HepG2 cells. HepG2 cells expressing PHB2-WTFlag were transduced with control (SRC-NC) or SRC-overexpressing lentivirus. IP-Western blot analysis as described in (B). E Effect of SRC knockdown on PHB2 phosphorylation in HepG2 cells. HepG2 cells expressing PHB2-WTFlag were transfected with scramble control or SRC-specific shRNA. IP-Western blot analysis as described in (B). F Identification of SRC phosphorylation sites on PHB2 in HepG2 cells. HepG2 cells were transfected with various Flag-tagged PHB2 mutants (Y34F, S39A, Y77F, Y34F/S39A, Y34F/Y77F, S39A/Y77F) with or without SRC overexpression. IP-Western blot analysis shows phosphorylation levels of different PHB2 mutants. All immunoprecipitation and Western blot experiments were performed in at least three independent experiments with similar results. Representative blots are shown. G The KM survival curve of the Src gene in TCGA data, where different groups are tested using the log-rank test. HR (High exp) represents the hazard ratio of the high expression group relative to the low expression group.