Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/elife.66095

Figure Lengend Snippet: Figure 2. Individual knockouts of the CRTC family members in human LKB1-null lung cancer cells inhibit the CREB-mediated target gene expression and moderately affect cell viability and anchorage-independent growth. (A) Western blot analysis of endogenous CRTC proteins in parental A549 cells, A549 cells stably transduced with non-targeting sgRNA, and two independent single knockout clones for each CRTC1, CRTC2, or CRTC3. The protein level of a CREB target gene, PDE4D was also detected. Blotting with anti-b-ACTIN was used as a loading control. (B) The transcript levels of CREB- mediated target genes (INSL4, LINC00473 and NR4A2) were determined by RT-qPCR assays (n = 2). The LKB1-wt cells, H522 parental (PA) cells, were also analyzed. (C,D) Individual CRTC knockout or control cells were cultured at 3 105 cells/well in the 6-well plates for 96 hr. The viable cells were quantified by trypan blue exclusion assay (C), and the number of apoptotic cells was determined by staining with annexin V/propidium iodide (PI) followed by flow cytometry (D). (E) Control and CRTC knockout cells were cultured in soft agar for 14 days, and the resulting colonies were stained by crystal violet and photographed under microscope. The number of colonies was counted using ImageJ. Assays were performed in triplicate. One-way ANOVA test was used to calculate the p values (*p<0.05, **p<0.01, ns p>0.05). The online version of this article includes the following source data and figure supplement(s) for figure 2:

Article Snippet: The control plasmid sgCtr-LentiCRISPRv2 expressing a non-target sgRNA (#107402) (Gao et al., 2017), lentiCas9-Blast (#52962) and lentiGuide-Puro constructs containing non-targeting control gRNA (#80180), STK11 gRNA-1 (#75912), and STK11 gRNA-2 (#75913) were also purchased from Addgene (Doench et al., 2016).

Techniques: Targeted Gene Expression, Western Blot, Stable Transfection, Transduction, Knock-Out, Clone Assay, Control, Quantitative RT-PCR, Cell Culture, Trypan Blue Exclusion Assay, Staining, Flow Cytometry, Microscopy