高分辨解析神经发育，深挖皮层神经元迁移调控机制

Fig. 1. PA-triggered B7H3 palmitoylation enhances B7H3 protein stability.
A Heatmap of B7 family immunohistochemical (IHC) staining score in MSI-H and MSS tumors. B Statistical results of IHC staining scores of B7 family in MSI-H and MSS tumors. (n?=?10). C RNA-seq of MSS and MSI-H tumor samples followed by Gene Ontology (GO) enrichment analysis of differentially expressed genes. D Heatmap of the top 50 representative metabolites in MSS and MSI-H tumor samples. E Kyoto encyclopedia of genes and genomes (KEGG) circos plots illustrating differential metabolites identified through untargeted metabolomics in MSS and MSI-H tumor samples. F KEGG-enriched pathway of metabolites with increased levels in MSS tumors compared to MSI-H. Immunoblot (IB) analysis of B7H3 in RKO cells treated with DMSO, glutaric acid (1?mM), palmitoylcarnitine (5?μM), hexadecanal (10?μM), palmitic acid (100?μM), or coenzyme A (3?mM) for 24?h (G) and quantification (H) (n?=?3). I Acyl-biotin exchange (ABE) assay of B7H3 palmitoylation in RKO or SW480 cells after PA treatment. PA palmitic acid. HAM hydroxylamine. The samples derive from the same experiment, but different gels for B7H3, and another for Palm-B7H3 were processed in parallel. J ABE assay of B7H3 palmitoylation in RKO or SW480 cells after 2-bromopalmitate (2-BP, 50?μM) treatment. The samples derive from the same experiment, but different gels for B7H3, and another for Palm-B7H3 were processed in parallel. K IB analysis of B7H3 after 2-BP treatment at indicated doses/times. IB analysis of B7H3 stability after CHX (100?μg/ml) treatment with 2-BP (50?μM) (L) and quantification (M) (n?=?3). Data indicate the mean?±?SD, by unpaired 2-tailed Student’s t test (B), One-way ANOVA with Dunnett’s multiple comparisons test (H), and two-way ANOVA with Tukey’s test (M). n?=?3 independent experiments (G, I–L). Source data are provided as a Source Data file.

Fig. 2. B7H3 C496 palmitoylation suppresses SQSTM1/p62-mediated autophagic degradation.
IB analysis of B7H3 after treatment with MG132 (10?μM), bafilomycin A1 (Baf A1, 0.2?μM), 3-methyladenine (3-MA, 5?mM), or chloroquine (CQ, 50?μM) for 8?h (A) and quantification (B). (n?=?3). IB analysis of B7H3 stability after CHX (100?μg/ml) treatment in WT and BECN1 KO RKO cells (C) and quantification (D). (n?=?3). E Co-immunoprecipitation (co-IP) of HA-B7H3 with indicated Flag-tagged cargo receptors in HEK293T cells. The samples derive from the same experiment, but different gels for Flag, and another for HA and GAPDH, were processed in parallel. IB analysis of B7H3 stability after CHX (100?μg/ml) treatment in WT and SQSTM1 KO RKO cells (F) and quantification (G). (n?=?3). H IB analysis of B7H3 in WT and SQSTM1 KO RKO cells treated with DMSO or 2-BP (100?μM). I Schematic of the predicted B7H3 palmitoylation site. J ABE assay of palmitoylation of B7H3 WT and indicated site mutants. The samples derive from the same experiment, but different gels for B7H3, and another for Palm-B7H3 were processed in parallel. K Multiple sequence alignment of B7H3 across species by Jalview. L Co-IP of Flag-SQSTM1 with HA-B7H3 WT or C496A in HEK293T cells. M ABE assay of palmitoylation in RKO stable cell lines expressing B7H3 WT or C496A. The samples derive from the same experiment, but different gels for B7H3, and another for Palm-B7H3 were processed in parallel. IB analysis of B7H3 stability after CHX (100?μg/ml) treatment in RKO^WT and RKO^C496A cells (N) and quantification (O). (n?=?3). P Representative images of confocal microscopy after treatment of RKO^WT or RKO^C496A with EBSS and CQ (50?μM) for 8?h. Scale bars, 5?μm. Data indicate the mean?±?SD, by unpaired 2-tailed Student’s t test (B) and two-way ANOVA with Tukey’s test (D, G, O). n?=?3 independent experiments (A, C, E, F, H, J, L–N, P). Source data are provided as a Source Data file.

Fig. 3. B7H3 palmitoylation inhibits the antitumor activity of CD8+ T cells.
Tumor growth (A) and tumor weight (B) of CT26^WT versus CT26^C278A tumors in BALB/c mice (n?=?5). Tumor growth (C) and tumor weight (D) of CT26^WT versus CT26^C278A tumors in NCG mice (n?=?5). Spectral flow cytometry analysis of CD45? immune infiltrates in CT26^WT and CT26^C278A tumors from BALB/c mice (n?=?5): UMAP clustering (E), immune composition (F), proportions of immune subpopulations (G), and CD8? T-cell density map (H). I–K C57BL/6J mice were intraperitoneally injected with 200?μg of anti-CD8α depleting antibody or isotype control per mouse one day before subcutaneous inoculation of MC38^WT or MC38^C278A cells, and then once weekly thereafter for a total of three doses (n?=?5). Schematic (I), tumor growth curves (J), and tumor weights (K). (L to R). MC38^KO, MC38^WT, and MC38^C278A cells were injected subcutaneously into C57BL/6?J mice (n?=?5). L Tumor growth curves. M Tumor weights. N Relative levels of tumor-infiltrating CD8+ T by flow cytometry. O Representative images of immunofluorescence of CD8+ T cells and CD45+ cells in subcutaneous graft tumors. Scale bars, 50?μm. P Quantification of CD8? cells among CD45? tumor-infiltrating leukocytes (TILs) from (O) (n?=?5). For each tumor, three fields were quantified and averaged to obtain one value per tumor. Q Flow cytometry analysis of Granzyme B (GZMB) expression in tumor-infiltrating CD8+ T cells. R Flow cytometry analysis of Interferon-γ (IFN-γ) expression in tumor-infiltrating CD8+ T cells. S, T RKO^KO, RKO^WT, and RKO^C496A cells were co-cultured with activated human T lymphocytes (n?=?5). S Statistical analysis shows the percentage of caspase-3 in tumor cells by flow cytometry. T Cell-free supernatants were collected, and LDH release was measured to calculate percent cytotoxicity. Data indicate the mean?±?SD, by unpaired 2-tailed Student’s t test (B, D), one-way ANOVA with Tukey’s test (K, M, N, P, Q, R), and two-way ANOVA with Tukey’s test (A, C, J, L, S, T). Source data are provided as a Source Data file.

Fig. 4. ZDHHC24-mediated B7H3 palmitoylation promotes protein stability and is linked to poor colorectal cancer outcomes.
A Co-IP showing endogenous B7H3–ZDHHC24 interaction. B Confocal images showing co-localization of B7H3 and ZDHHC24 in RKO cells. Scale bars, 5?μm. C ABE assay of B7H3 palmitoylation in ZDHHC24-knockdown RKO cells. IB of B7H3 in RKO (D) and MC38 (E) cells with control or ZDHHC24/Zdhhc24 knockdown. F Confocal images of B7H3 staining in RKO shNC or shZDHHC24 cells. Scale bars, 5?μm. IB of B7H3 after CHX (100?μg/mL) for the indicated times (G) and quantification (H) (n?=?3). IB of B7H3 protein levels (I) and ABE assay of B7H3 palmitoylation (J) after ZDHHC24 overexpression in RKO^{B7H3 WT} and RKO^{B7H3 C496A} cells. K Co-IP of B7H3 with SQSTM1 in RKO^{B7H3 WT} and RKO^{B7H3 C496A} cells with or without Flag-ZDHHC24 expression. L Confocal images of RKO shNC or shZDHHC24 cells after EBSS and CQ (50?μM) treatment for 8?h. Scale bars, 5?μm. M Duolink PLA showing B7H3–p62 interactions. Scale bars, 5?μm. N Kaplan–Meier overall survival stratified by ZDHHC24 expression (n?=?94; log-rank test). O ZDHHC24 IHC staining in paired adjacent and primary tissues (n?=?86 pairs). P ZDHHC24 IHC staining scores across tumor stages (n?=?94). Q Association between ZDHHC24 and B7H3 IHC staining (n?=?94). The samples (D, E, G, I, K) derive from the same experiment, but different gels for ZDHHC24/Flag, and another for B7H3 and GAPDH were processed in parallel. The samples (C, J) derive from the same experiment, but different gels for ZDHHC24, another for GAPDH, another for B7H3, and another for Palm-B7H3 were processed in parallel. Data indicate the mean?±?SD, by two-way ANOVA with Tukey’s test (H), paired 2-tailed Student’s t test (O), chi-square test (P), and two-sided chi-square test (Q). n?=?3 independent experiments (A–G, I–M). Source data are provided as a Source Data file.

Fig. 5. ZDHHC24 promotes tumor growth by inhibiting the antitumor activity of CD8+ T cells.
A Schematic of azoxymethane (AOM)/ dextran sulfate sodium (DSS)-induced colitis-associated colorectal cancer (CAC) model. Body weight change (B) and colon length (C) of Zdhhc24^fl/fl and Zdhhc24^ΔIEC mice during AOM/DSS treatments (n?=?8). D Representative images of Zdhhc24^fl/fl and Zdhhc24^ΔIEC mice after longitudinal sectioning of the colon. Tumor number (E) and tumor size distributions (F, G) in Zdhhc24^fl/fl and Zdhhc24^ΔIEC mice (n?=?8). Representative H&E staining (H) and immunofluorescence staining (I) of colon tissues/tumors in Zdhhc24^fl/fl and Zdhhc24^ΔIEC mice. Scale bars, 0.25?mm (H), 400?μm (top in I), and 50?μm (bottom in I). J–N MC38 shNC, MC38 shZdhhc24 (#1 and #2) cells were injected subcutaneously into C57BL/6J mice (n?=?5). J Tumor growth curves. K Tumor weights. L Relative levels of tumor-infiltrating CD8+ T by flow cytometry. M Flow cytometry analysis of GZMB expression in tumor-infiltrating CD8+ T cells. N Flow cytometry analysis of IFN-γ expression in tumor-infiltrating CD8+ T cells. O Quantification of CD8+T cell percentage in CRC tissue microarrays stratified by ZDHHC24 IHC expression (ZDHHC24^Low, n?=?48; ZDHHC24^High, n?=?46). Violin plots show the data distribution. Box plots show the median (center marker), the 25th and 75th percentiles (box limits) and whiskers extending to 1.5?×?IQR; points beyond the whiskers represent outliers. P Correlation of ZDHHC24, B7H3 and CD8? T cell IHC staining scores in CRC tissue microarrays (n?=?94) assessed by Pearson’s correlation (two-tailed); r and exact P values are shown (no adjustment for multiple comparisons was applied). Data indicate the mean?±?SD, by unpaired 2-tailed Student’s t test (B, C, E, F, O), one-way ANOVA with Tukey’s test (K, L, M, N). Two-way ANOVA with Tukey’s test (J). n?=?3 independent experiments (H, I). Source data are provided as a Source Data file.

Fig. 6. ZDHHC24 inhibition of antitumor effects on CD8+ T cells requires B7H3 palmitoylation.
A–I MC38^WT+Vector, MC38^WT+Zdhhc24, MC38^C278A+Vector, and MC38^C278A+ Zdhhc24 cells were injected subcutaneously into C57BL/6J mice (n?=?5). A The volume of the subcutaneous graft tumors was measured on the indicated days, and growth curves were drawn. B Quantification of measured weights of subcutaneous graft tumors. C Relative levels of tumor-infiltrating CD8+ T cells by flow cytometry. D Representative immunofluorescence images of tumor tissues. Scale bars, 50?μm. E Quantification of CD8?/CD45? TILs from (D) (n?=?5). For each tumor, three fields were quantified and averaged to obtain one value per tumor. F Flow cytometry analysis of IFN-γ expression in tumor-infiltrating CD8+ T cells. G Representative images of tumor-infiltrating IFN-γ+CD8+ T cells analyzed by flow cytometry. H Flow cytometry analysis of GZMB expression in tumor-infiltrating CD8+ T cells. I Representative images of tumor-infiltrating GZMB+ CD8+ T cells analyzed by flow cytometry. J, K RKO^WT +?Vector, RKO^WT +?ZDHHC24, RKO^C496A +?Vector, and RKO^C496A +?ZDHHC24 cells were co-cultured with activated human T lymphocytes (n?=?5). J Statistical analysis shows the percentage of caspase-3+ in tumor cells by flow cytometry. K Cell-free supernatants were collected, and LDH release was measured to calculate percent cytotoxicity. Data indicate the mean?±?SD, by one-way ANOVA with Tukey’s test (B, C, E, F, H) and two-way ANOVA with Tukey’s test (A, J, K). Source data are provided as a Source Data file.