Fig. 2: The proliferative potential of HKCs and SCC cells is suppressed by ULK3 downmodulation. | Nature Communications

Fig. 2: The proliferative potential of HKCs and SCC cells is suppressed by ULK3 downmodulation.

From: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

Fig. 2

a Cell growth on days 3 and 5 of HKC (strain #TP26) and of three SCC cell lines infected with two ULK3 silencing lentiviruses (shULK3 #1, #2) versus empty control vector (shCTR) plated in triplicate at low-density. Representative experiment of two independent biological replicates. The efficient downregulation of ULK3 in HKCs and SCC cells is in Supplementary Fig. 2a, b. b Live fluorescence images of SCC13 and Cal27 cells infected with two ULK3 silencing lentiviruses, or control virus, and stained for early- (phosphatidylserine-FITC, green), late- apoptosis (7-aminoactinomycin D, magenta) and for all viable cells (CytoCalcein, violet-blue). Quantification, in duplicate cultures, SCC13 n(fields/shCTR) = 6, n(fields/shULK3) = 25; Cal27 n(fields/shCTR) = 7, n(fields/shULK3) = 24, n(cells/field) > 90, mean ± SEM, P < 0.001 or P < 0.0001 one-way ANOVA. Scale bar 10 µm. c Colony formation of HKCs (strain # TP26), five SCC cell lines and HeLa cells infected with two ULK3 silencing lentiviruses, or a control virus, and plated at limited cell density for one week. Crystal violet stained dishes were quantified using ImageJ. n(dishes) = 3, mean ± SEM, P < 0.0001, one-way ANOVA. Representative images of the stained colonies are in Supplementary Fig. 2c. Supplementary Fig. 2d-e show similar assays with SCC cells treated with the ULK3 inhibitor SU666819. d Surveyor Assay41 of SCC13 cells one week after the infection with lentiviruses co-expressing the Cas9 enzyme and one of the three RNA guides targeting ULK3 (gRNA ULK3 #1-3), or a scrambled control gRNA (SCR gRNA). Each targeted genomic region (amplified using corresponding oligo pairs (#1-3) (-)) was tested for DNA mismatch by PCR by incubation with single-strand DNAse. (+). In control reactions, we used genomic DNA from SCR-infected SCC13 cells. The arrows indicate the amplified region and the asterisks the DNAase cleavage products. e ULK3 and ß-ACTIN immunoblotting of SCC13 cells one week after the infection with ULK3 gRNA (#1-3), versus a scrambled gRNA (SCR) lentiviruses. f Colony formation of SCC13 cells, plus/minus ULK3 deletion as above, plated at limited dilution for six days. n(dishes) = 3, mean ± SEM, P < 0.0001, one-way ANOVA.

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