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. 2021 Nov 16;41(4):538–549. doi: 10.1038/s41388-021-02114-8

Fig. 6. CBX3 binds to TRIM28, TRIM24, or RBBP4 to regulate ARHGAP24 expression in LUAD cells.

Fig. 6

a PPI network between CBX3 and proteins correlated with CBX3. b The whole cell lysates (WCL) of A549 were collected to undergo immunoprecipitation by using the IgG and CBX3 antibodies. Western blotting analysis was using used to detect the RBBP4, TRIM24, TRIM28 and CBX3. c–e The whole cell lysates (WCL) of A549 were collected to undergo immunoprecipitation by using the IgG and TRIM28, TRIM2, or RBBP4 antibodies, respectively. Western blotting analysis was using used to detect the RBBP4, TRIM24, TRIM28 and CBX3. f, g A549 cells were infected with shControl, shTRIM28 #1, or shTRIM28 #2 for 72 h. Cells were collected for Western blotting analysis (f) and RT-qPCR analysis (g). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. *P < 0.05; **P < 0.01; ***P < 0.001. h, i A549 cells were infected with shControl, shTRIM24 #1, or shTRIM24 #2 for 72 h. Cells were collected for Western blotting analysis (h) and RT-qPCR analysis (i). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. *P < 0.05; **P < 0.01; ***P < 0.001. j, k A549 cells were infected with shControl, shRBBP4 #1, or shRBBP4 #2 for 72 h. Cells were collected for Western blotting analysis (j) and RT-qPCR analysis (k). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. *P < 0.05; **P < 0.01; ***P < 0.001. l The ChIP-qPCR of TRIM28 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates (n = 3). NS not significant; ***P < 0.001. Primer I indicated the pair of primer located in the common binding peak of RBBP4, TRIM24, TRIM28, CBX3 and H3K9me3; Primer O indicated the the pair of primer located outside the common binding peak of RBBP4, TRIM24, TRIM28, CBX3 and H3K9me3. m The ChIP-qPCR of TRIM24 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates (n = 3). NS not significant; ***P < 0.001. n The ChIP-qPCR of RBBP4 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates (n = 3). NS not significant; **P < 0.01. o A549 cells were infected with shControl and shTRIM28 for 72 h. Cells were collected for the ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates (n = 3). *P < 0.05; **P < 0.01. p A549 cells were infected with shControl and shTRIM24 for 72 h. Cells were collected for the ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. ***P < 0.001. q A549 cells were infected with shControl and shRBBP4 for 72 h. Cells were collected for the ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. *P < 0.05; **P < 0.01; ***P < 0.001. r–t A549 cells were infected with indicated shRNAs for 72 h. Cells were harvested for Western blotting analysis.