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

Fig. 3. CBX3 increases the amount of active Rac1 in LUAD.

Fig. 3

a, b GSEA for CBX3 in TCGA-LUAD (a) and GSE68465 (b) datasets. Samples were first divided into two groups according to the median expression level of the CBX3. Then, differential expression analysis was applied between the high and low expression groups. Input genes for GSEA were sorted by their logFC values. Signaling pathways activated or suppressed by CBX3 were decided by the NES value derived from GSEA. c, d Rho GTPase signaling pathway was activated by the over expression of CBX3 in single cells of A459 (c) and LC-2/ad (d). Median expression level of CBX3 was used as cutoff. e, f Knockdown of CBX3 in A549 cell line leaded to the suppression of Rho GTPases signaling pathway. g Key genes involved in the regulation of Rho GTPases signaling pathway by the knockdown of CBX3 in A459 cell line. h, i A459 and H1299 cells were infected with shConrtol or shCBX3 for 72 h. Cells were divided in to two equal parts. The first part of cells was collected for Western blotting analysis and detected by the CBX3, Rac1 and Beta-actin antibodies. The second part of cells were lysed and subjected to GST-pull down following the protocol of Active Rac1 Detection Kit, the active Rac1 was detected by Western blotting analysis. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with two replicates (n = 2). *P < 0.05. j, k A459 and H1299 cells were transfected with indicated constructs for 24 h. Cells were divided in to two equal parts. The first part of cells was collected for Western blotting analysis and detected by the CBX3, Rac1 and Beta-actin antibodies. The second part of cells were lysed and subjected to GST-pull down following the protocol of Active Rac1 Detection Kit, the active Rac1 was detected by Western blotting analysis. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with two replicates (n = 2). *P < 0.05; **P < 0.01; ***P < 0.001.