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. 2022 Nov 15;132(22):e161308. doi: 10.1172/JCI161308

Figure 1. Mutant KRAS promotes BCL6 expression.

Figure 1

(A and B) mRNA (A) and protein (B) expression levels of BCL6 in normal lung (N) and tumor tissue (T) from LSL-KrasG12D/+ mice (n = 3). LSL-KrasG12D mice were infected with intranasal Ad-Cre for 16 weeks. (C and D) mRNA (C) and protein (D) expression levels of BCL6 in MEFs. MEFs were isolated from LSL-KrasG12D mice and infected with Ad-Cre for 72 hours (n = 3). (E) Correlation analysis. Correlation between BCL6 and KRAS-GTP protein expression levels in different cancer cell lines (n = 20). KRAS-GTP levels were determined using GST-RBD, the GST-fusion of the RAS binding domain of C-RAF, to pulldown active GTP-bound RAS from cellular lysates by glutathione beads. R, Pearson’s correlation coefficient. (F and G) KRAS mutant variants upregulated BCL6 expression at the mRNA (F) and protein (G) levels. 293T cells were transfected with different doxycycline-inducible G12 KRAS mutant variants: pLVX-TetOne-KRAS(G12C), pLVX-TetOne-KRAS(G12D), pLVX-TetOne-KRAS(G12S), pLVX-TetOne-KRAS(G12V). Transfected cells were then treated with or without 2 μM doxycycline (Dox) for 96 hours. KRAS activity was determined using GST-RAF-RBD to pulldown GTP-KRAS from cell lysates. (H) Heatmap showing differentially expressed BCL6 target genes (P < 0.05) in HPNE and HPNE/KRAS cells (n = 3). z score was calculated based on counts of exon model per million mapped reads. (I) The mRNA expression levels of BCL6 target gene in HPNE and HPNE/KRAS cells (n = 3). Data in A, C, F, and I are expressed as mean ± SEM of 3 technical replicates, representative of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, determined via unpaired 2-tailed Student’s t test. The immunoblots in B, D, and G were contemporaneous and run in parallel from the same biological replicate.