Fig 2: ZDHHC5 promotes GBM development in vivo. A and B Luciferase-expressing ZDHHC5-knockdown U251 and T98G cells were injected into athymic nude mice (n = 10). The mice were subjected to IVIS scanning on days 7,14,21,28 after the injection of tumor cells. Representative bioluminescent images of intracranial GBM xenografts are shown on the left. Quantification of bioluminescent images (day = 28) is shown on the right. C and D Kaplan‒Meier survival curves of mice intracranially injected with U251 and T98G cells with the indicated modifications (n = 10). P values were calculated using the two-tailed Student's t test for (A, B) and the log-rank (Mantel‒Cox) test for (C, D). **P < 0.01, ***P < 0.001, ****P < 0.0001. E and F IHC images of the indicated proteins are shown in consecutive brain sections. Scale bars, 20 µm

Fig 3: ZDHHC5-mediated FAK S-palmitoylation promotes cell proliferation, cell invasion and EMT in vitro. A IB for ZDHHC5 in U251 and T98G cells transfected with ZDHHC5 shRNAs or shRNA control. B CCK8 assays of U251 and T98G cells transfected with ZDHHC5 shRNAs or shRNA control. C Colony formation assay of U251 and T98G cells transfected with ZDHHC5 shRNAs or shRNA control. D Wound-healing assay of ZDHHC5 shRNAs or shRNA control. scale bars: 25 μm. E Invasion of U251 and T98G cells transfected with ZDHHC5 shRNAs or shRNA control; scale bars: 100 μm. (F) IB for the indicated proteins in U251 and T98G cells transfected with ZDHHC5 shRNAs or shRNA control. Data are represented as the mean ± SD (n = 3). Statistical analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig 4: FAK is palmitoylated at Cys456 (A) U251 cells were cotransfected with Flag-FAK WT or the indicated mutants and/or Myc-ZDHHC5. The cells were metabolically labeled with 50 µM alkynyl PA for 4 h. The levels of FAK palmitoylation were analyzed by performing a click reaction and streptavidin bead pulldown, followed by IB. B Amino acid sequences around the cysteine 456 residue of the FAK protein across different species. C and D U251 and T98G cells were transfected with Flag-FAK WT or Flag-FAK C456S and then metabolically labeled with 50 M alkynyl PA for 4 h. FAK palmitoylation levels were assessed by click reaction and streptavidin bead pulldown, followed by IB. E U251 and T98G cells were transfected with Flag-FAK WT or Flag-FAK C456S. The APE assay was conducted to analyze FAK palmitoylation in U251 cells with the specified modifications. F and G U251 and T98G cells were transfected with Flag-FAK WT or Flag-FAK C456S. The levels of FAK in the membrane and cytoplasmic fractions were evaluated using IB with the indicated antibodies. H and I U251 and T98G cells were transfected with Flag-FAK WT or Flag-FAK C456S. Then, IB was performed with the indicated antibodies

Fig 5: ZDHHC5 regulates FAK S-palmitoylation by directly binding to FAK (A) U251 cells infected with siRNAs targeting different zDHHC proteins. The cells were metabolically labeled with alkynyl PA (50 µM) for 4 h. FAK palmitoylation levels were assessed by click reaction and streptavidin bead pulldown, followed by IB. B Cell lysates from U251 and T98G cells were analyzed by IP using antibodies against FAK and ZDHHC5 and then subjected to IB analysis. IgG was used as the isotype control. C HEK293T cells transfected with Flag-FAK and/or Myc-ZDHHC5 were subjected to IP-IB and IB using the indicated antibodies. D In vitro protein binding assays with purified ZDHHC5 and FAK protein. The purities of ZDHHC5 and FAK were examined by SDS-PAGE and Coomassie Blue Staining. E Schematic representations of ZDHHC5, FAK, and their shortened mutants. F HEK293T cells were cotransfected with Flag-FAK and FL Myc-ZDHHC5 or its deletion mutants. The cell lysates were subjected to Flag bead pulldown, followed by IB using specific antibodies. G HEK293T cells were cotransfected with Myc-ZDHHC5 and FL Flag-FAK or its deletion mutants. Cell lysates were analyzed by Flag bead pulldown, followed by IB using the indicated antibodies