Fig 2: Effect of MYO6 knockdown on cell apoptosis.(A) Cytogram of Annexin V-APC binding vs. 7-AAD uptake in U251 cells following lentivirus infection in shCon and shMYO6 groups. (B) Quantification of apoptotic cells in U251 cells by FACS. Annexin V+/7-AAD-: early apoptotic cells; Annexin V+/7-AAD+: late apoptotic cells; Annexin V-/7-AAD-: viable cells; Annexin V-/7-AAD+: cells in necrosis.

Fig 3: Cellular analysis of novel MYO6 variant. (A) Western blot of whole-cell lysates and membrane proteins from HeLa cells transfected with plasmids carrying human MYO6WT and MYO6p.E60Q. (B) Quantification ratio of the normalized integrated density of MYO6 expression from membrane/whole proteins. (C) Representative immunofluorescence staining images of HeLa cells labeled by MYO6 (red), pan-cadherin (green), and DAPI (blue). (D) Quantification of the relative fluorescence intensity of MYO6. (E) Quantification of the ratio of the normalized relative fluorescence intensity of MYO6/pan-cadherin. All values are represented as the mean ± SEM from independent experiments. Statistically significant differences were assessed by (B) unpaired t-test, * p < 0.05 or (D,E) one-way ANOVA followed by Tukey’s multiple comparisons, * p < 0.05; *** p = 0.0001; **** p < 0.0001.

Fig 4: Mutation analysis of c.178G>C MYO6 variant. (A) Sanger sequence of heterozygous and homozygous missense c.178G>C variant in exon 3 of MYO6. (B) Schematic diagram of myosin VI protein. (C) MYO6 ontology across multiple species highlighting the conserved E60 residue.

Fig 5: Knockdown of MYO6 inhibits viability and proliferation of glioblastoma U251 cells.(A) MTT showing growth curves determined in U251 cells. The number of viable cells was much fewer in the shMYO6 group than in the shCon group. (B) Representative colony formation showing clonogenic survival determined in U251 cells. (C) The number of colonies was much fewer in the shMYO6 group than in the shCon group. Data are mean±SD (n=3; t-test). *p<0.001; scale bar, 250 µm.