Journal: Cancer Gene Therapy

Article Title: ZDHHC1 downregulates LIPG and inhibits colorectal cancer growth via IGF2BP1 Palmitoylation

doi: 10.1038/s41417-024-00808-1

Figure Lengend Snippet: The expression of ZDHHC family members in CRC were presented by box-plot analyses ( A ). Three-years overall CRC survival of ZDHHC1, ZDHHC3 and ZDHHC11 were analyzed via TIMER 2.0 ( B – D ). Cells were harvested for WB analysis ( E – G ). HCT116 and SW480 cell growth for 48 h in complete medium: medium containing 10% FBS; lipoprotein-free medium: medium containing 10% free lipoprotein FBS ( H – J ). Data presented as mean ± SD with three replicates. Student’s t -test was used to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used were ZDHHC1 (26545-1-AP; Proteintech; 1:300 dilution) and LIPG (67434-1-lg; Proteintech; 1:300 dilution).

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: ZDHHC1 downregulates LIPG and inhibits colorectal cancer growth via IGF2BP1 Palmitoylation

doi: 10.1038/s41417-024-00808-1

Figure Lengend Snippet: HCT116 cells were subjected to RNA-seq, subsequent KEGG pathway enrichment ( A ) and volcano plots analysis ( B ). Cells with knock-down ( C ) and overexpression ( D ) of ZDHHC1were harvested for western blotting and RT-qPCR analysis. The typical IHC images of CRC tissue microarray stained with ZDHHC1 and LIPG ( E ). The size of the scale bar on microscopy images as indicated in the figure. The correlation analysis between ZDHHC1 and LIPG in CRC tissues ( F ). Pearson correlation was used to determine statistical significance; the P -value was indicated in the figure. Western blot and RT-PCR were used to analyze LIPG protein and mRNA expression ( G ). HCT116 cells were incubated for 48 h with 800 µg HDL in serum-free DMEM and no substrate with or without GSK (32 nM) ( H ). Lipid droplets were visualized with Bodipy 493/503 staining (green). Nuclei were stained with DAPI (blue). Scale bar, 40 μm. Intracellular triacylglyceride (TAG) levels were quantified with the triglyceride quantification assay kit ( I ). Data presented as mean ± SD with three replicates. Student’s t- test and one-way ANOVA were used to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used were ZDHHC1 (26545-1-AP; Proteintech; 1:300 dilution) and LIPG (67434-1-lg; Proteintech; 1:300 dilution).

Techniques: RNA Sequencing, Knockdown, Over Expression, Western Blot, Quantitative RT-PCR, Microarray, Staining, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

Journal: Cancer Gene Therapy

Article Title: ZDHHC1 downregulates LIPG and inhibits colorectal cancer growth via IGF2BP1 Palmitoylation

doi: 10.1038/s41417-024-00808-1

Figure Lengend Snippet: Venn diagram showed IGF2BP1 and TARDBP were the potential binding partners of ZDHHC1 ( A ). Co immunoprecipitation analysed and verified the interaction between ZDHHC1 and IGF2BP1 in HCT116 and SW480 cells ( B , C ). IGF2BP1 knockdown decreased the protein and mRNA level of LIPG by western blotting and RT-qPCR analysis ( D ). RIP-qPCR analysis showing the enrichment of LIPG mRNA in anti-IGF2BP1 precipitates ( E ). MeRIP-qPCR analysis showing the m6A enrichment of LIPG mRNA in HCT116 and SW480 cells ( F ). Two very high-confidence m6A site was identified upon LIPG mRNA based on the SRAMP software analysis ( G ). Schematic representation of LIPG- WT (wild-type) or LIPG- MT (mutated-type) sequence ( H ). Luciferase reporter assays measured the luciferase activities of LIPG WT or LIPG Mut in CRC cells with IGF2BP1 knockdown ( I ). After silencing IGF2BP2 in HCT116 and SW480 cells, the mRNA half-lives and expression of LIPG were analyzed at the predetermined times following actinomycin D (5 μg/ml) treatment ( J , K ). Data presented as mean ± SD with three replicates. Student’s t -test was used to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used were ZDHHC1 (26545-1-AP; Proteintech; 1:300 dilution) and LIPG (67434-1-lg; Proteintech; 1:300 dilution).

Techniques: Binding Assay, Immunoprecipitation, Knockdown, Western Blot, Quantitative RT-PCR, Software, Sequencing, Luciferase, Expressing

Journal: Cancer Gene Therapy

Article Title: ZDHHC1 downregulates LIPG and inhibits colorectal cancer growth via IGF2BP1 Palmitoylation

doi: 10.1038/s41417-024-00808-1

Figure Lengend Snippet: ZDHHC1 could not regulate the protein and mRNA level of IGF2BP1 by western blotting and RT-qPCR analysis ( A , B ). IP-ABE analysis of IGF2BP1 palmitoylation in HCT116 cells when treated with or without palmitoylation inhibitor 2-BP ( C , D ). Predicted cysteine residues on IGF2BP1 susceptible to S-palmitoylation ( E ). IP-ABE analysis of IGF2BP1 palmitoylation in HCT116 cells when ectopically expressing IGF2BP1-WT or harboring specific mutations ( F ). Alignment of the similarity of PCSK9 sequences in different vertebrate orthologs ( G ). Flga-ZDHHC1 reduce the level of LIPG with IGF2BP1-WT but not IGF2BP1-C337S by western blotting analysis ( H ). RIP-qPCR analysis showing the enrichment of LIPG mRNA in anti-IGF2BP1 precipitates ( I ). MeRIP-qPCR analysis showing the m6A enrichment of LIPG mRNA in HCT116 cells ( J ). Data presented as mean ± SD with three replicates. Student’s t- test and one-way ANOVA were used to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used were ZDHHC1 (26545-1-AP; Proteintech; 1:300 dilution) and LIPG (67434-1-lg; Proteintech; 1:300 dilution).

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: Cancer Gene Therapy

Article Title: ZDHHC1 downregulates LIPG and inhibits colorectal cancer growth via IGF2BP1 Palmitoylation

doi: 10.1038/s41417-024-00808-1

Figure Lengend Snippet: HCT116 cells were infected with indicated shRNAs and harvested for western blotting analysis and RT-qPCR analysis ( A ). HCT116 cells were infected with indicated shRNAs and plasmids, then harvested for western blotting analysis and RT-qPCR analysis ( B ). A graphic illustration of the proposed mechanism in this study ( C ). In brief, IGF2BP1 is palmitoylated by ZDHHC1, and then bound to the m6A site upon LIPG mRNA to reduce the stability and expression of LIPG mRNA, thereby inhibiting CRC cells from taking HDL, and leading to the decrease of CRC cell growth. Data presented as mean ± SD with three replicates. One-way ANOVA was used to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used were ZDHHC1 (26545-1-AP; Proteintech; 1:300 dilution) and LIPG (67434-1-lg; Proteintech; 1:300 dilution).

Techniques: Infection, Western Blot, Quantitative RT-PCR, Expressing