ccnl2  (Novus Biologicals)

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Boxplots of growth phenotypes for PC9-Cas9-mCherry cells expressing the indicated pgRNA compared to PC9-Cas9-GFP cells expressing a double-safe-targeting control pgRNA. Boxes indicate mean ± SEM of six biological replicates, which are shown as overlaid points. Growth phenotype is defined as the log 2 -scaled ratio of mCherry:GFP cell counts at the late time point compared to the day 1 mCherry:GFP cell counts. Expected DKO phenotypes are the sum of single KO growth phenotypes. The expected and observed DKO phenotypes were compared using a one-tailed t test. Data shown are for the time point with the most extreme difference between expected and observed DKO growth phenotypes, termed the late time point: CCNL1/CCNL2 (day 12), CDK4/CDK6 (day 7), MEK1/MEK2 (day 11), and OXSR1/STK39 (day 10). Full time course data are shown in . (B) Fluorescence microscopy images of competitive fitness assays on early (day 1) and late time points as indicated above for (A). Scale bar, 100 μM. (C) Western blot validation of single KO and DKO pgRNA-induced gene inactivation. For CCNL1, pie charts of percent mutant alleles based on next-generation sequencing are shown due to lack of a suitable CCNL1 antibody for western blotting. Additional genomic DNA-level validation data are presented in . See also and and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Expressing, Control, One-tailed Test, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Mutagenesis, Next-Generation Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (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 . 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 log 2 (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 and , , and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Two Tailed Test, CRISPR, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Software