Fig 2: Identification of cell-line-specific and shared synthetic lethal paralog pairs(A) Rank plot of target-level GI scores in HeLa cells. Table insert, top synthetic lethal paralogs based on GI score.(B) Volcano plot of target-level GI scores in HeLa cells. FDR indicates the multiple hypothesis-adjusted p values from a two-tailed t test (Method details). Blue, synthetic lethal paralog GIs with GI < −0.5 and FDR < 0.1; red, buffering paralog GIs with GI > 0.25 and FDR < 0.1.(C) Scatterplot of target-level GI scores for paralog pairs in PC9 versus HeLa cells. Blue, synthetic lethal paralog pairs with GI < −0.5 and FDR < 0.1 in either PC9 or HeLa cells; gray, all paralog pairs with GI ≥ −0.5 or FDR ≥ 0.1.(D) CRISPR scores for representative synthetic lethal paralog pairs identified in the PC9 and HeLa cell screens. Top row: data shown are the mean CRISPR score for each single KO or DKO target across three biological replicates with replicate data shown in overlaid points. Shared synthetic lethal paralogs (e.g., CCNL1/CCNL2 and MEK1/MEK2) have FDR < 0.1 in both cell lines; PC9-specific paralogs (e.g., CDK4/CDK6 and OXSR1/STK39) have FDR < 0.1 in PC9 only; and HeLa-specific paralogs (e.g., GFTP1/GFPT2 and SOS1/SOS2) have FDR < 0.1 in HeLa only. Dashed lines indicate CRISPR score < −0.5. Bottom row: paralog gene expression in PC9 and HeLa cells from RNA-seq analysis. Dashed lines indicate log2(TPM) = 1, the threshold for gene expression.(E) Boxplots comparing the effect of CRISPR-mediated KO of the indicated gene in DepMap cell lines with high (top quartile) compared to low (bottom quartile) copy number of its paralogous gene. For boxplots, the middle line, hinges, notches, and whiskers indicate the median, 25th/75th percentiles, 95% confidence interval, and data points within 1.5× the interquartile range from the hinge, respectively. p values were computed using a two-tailed Wilcoxon rank-sum test. CRISPR score and copy number data were obtained from DepMap.(F) As in (E), but for gene expression.(G) Bar plot indicating the p values (computed using a two-tailed Wilcoxon rank-sum test) obtained by comparing the effect of a single paralog KO to the copy number (as in E) or gene expression (as in F) of its pair across human cancer cell lines profiled by DepMap. Bar color indicates whether each pair was synthetic lethal in PC9 only, HeLa only, or both cell lines in the pgPEN screens. Dashed line indicates p = 0.05.See also Figure S5 and Tables S4, S6, and S7.

Fig 3: CDK6 is a direct target of miR-1263. (A) Schematic illustration exhibiting overlapping of the target mRNAs of miR-1263 predicted by the DIANA, miRmap, miRwalk, and Targetscan databases. (B, C) Expression of predicted target genes in LM3 and97H cells transfected with miR-1263 mimics, an inhibitor, and corresponding NC. (D) Schematic illustration of the WT and MUT CDK6 sequences at the miR-1263 binding site. (E, F) The effects of miR-1263 mimics and inhibitor on the luciferase activities of the WT and MUT CDK6 mRNA 3’-untranslated regions as detected by qRT-PCR. (G) The effects of miR-1263 on the protein expression of TDO2 were detected by western blot analysis. (H, I) Western blotting showing CDK6 protein expression in transfected 97H and LM3 cells. (J, K) QRT-PCR showing CDK6 mRNA expression in transfected 97H and LM3 cells. (L) Relative CDK6 expression in 44 pairs of fresh frozen HCC tissues and matched normal liver tissues. (M) An obvious negative correlation between the levels of miR-1263 and CDK6 in 44 pairs of fresh frozen HCC tissues and matched normal liver tissues as determined by Pearson correlation analysis. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 4: The contribution of TH12-10 to the cell cycle. (A) Western blotting shows Cyclin D1, CDK4, CDK6, and p-Rb levels in NCM460 cells treated with TH12-10 at indicated concentrations for 48 h. (B) Densitometry analysis of indicated proteins as described in (A) was conducted and presented. (C) Flow cytometric analysis for cell-cycle distribution in NCM460 cells treated with TH12-10 or vehicle and then with nocodazole for a total of 48 h. (D) Quantitative analysis of NCM460 cells in G1 phase as described in (C). All data are presented as means ± SD of three independent experiments. N.S., not significant. Differences were tested using one-way ANOVA (B) or the unpaired t-test with Welch’s correction (D).

Fig 5: The establishment of ribociclib-resistant cell lines (RIBR).(A) EDR1 was established from MCF7-E10 cell lines by long-term culture with a steroid-depleted medium. MCF7-E10 cell lines were derived from MCF-7, which had been stably transfected with an ERE-GFP reporter plasmid, and monitored ER expression. EDR1 exhibited ER overexpression. RIBR was established from EDR1 after long-term (7 months) culturing with 1000 nM ribociclib in phenol red–free RPMI medium. (B) Cell proliferation of EDR1 and RIBR1,2 treated with ribociclib for 3 days was measured relative to the negative control. The results are expressed as mean ± SD of three independent experiments; * P < 0.05. (C) Colony formation assay of EDR1 and RIBR1,2 cell lines. The control (left) was cultured for 15 days without any drugs. Ribociclib (right) was harvested for 15 days with 1000 nM ribociclib. (D), EDR1 and RIBR1,2 cell lines were treated with DMSO or ribociclib (1000 nM) for 24 h and measured by the fluorescence-activated cell sorting (FACS) cell-cycle analysis. (E), the protein expression levels of CDK6 in EDR1, EDR1 with 1000 nM ribociclib for 24 h, and RIBR1,2 cell lines were analyzed by western blotting.