Journal: Cancer Research

Article Title: Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-21-3713

Figure Lengend Snippet: Figure 2. Depletion of KPNB1 reduces nuclear ASCL1 and ASCL1 downstream target gene expression in SCLC-A. A, Western blot showing whole-cell KPNB1 expression in SCLC-A subtype NCI-H2107 cells after transfection with control or KPNB1 siRNAs (siCtrl or siKPNB1). Western blots showing the level of ASCL1 in the nuclear (B) and whole-cell lysate (C) fractions of NCI-H2107 cells at 24 and 48 hours after siRNA transfection. Note, the same membranes were used for A and C as reflected by the GAPDH blots. D, Quantification of relative ASCL1 protein in the nuclear fractions of siCtrl versus siKPNB1-transfected cells across two independent experiments. Lamin B1, nuclear loading control (error bars, SEM; unpaired t test; , P ≤0.05). E, Comparison of ASCL1 downstream target expression by RT- qPCR in siCtrl (black)-, siKPNB1 (red)-, and siASCL1 (green)-transfected NCI-H2107 cells at 72 hours after transfection. Average of three technical replicates is shown (error bars, SEM, unpaired t test; , P ≤0.05; , P ≤0.01; , P ≤0.001; , P ≤0.0001; ns, nonsignificant). F, Diagram of CRISPRi lentiviral construct where the UbC promoter drives expression of catalytically inactive Cas9 (dCas9) fused to the KRAB transcriptional repressor. U6 promoter drives expression of gRNAs targeting the 50UTR of KPNB1 (gKPNB1.1, gKPNB1.2) or ASCL1 (gASCL1.1, gASCL1.2). G, Expression of dCas9-KRAB plus gKPNB1.1 or gKPNB1.2 resulted in an 84% reduction in KPNB1 protein. H,Western blot showing the level of ASCL1 in the nuclearfractionof NCI-H2107 cells expressing gKPNB1.1 or gKPNB1.2 versus control (Ctrl). I,Quantification of nuclear ASCL1 protein. J, Western blot showing undetectable levels of ASCL1 in SCLC-A subtype NCI-H2107 cells expressing dCas9-KRAB plus ASCL1.1 or ASCL1.2 gRNAs. K, Heatmap comparing expression of all DEG identified in CRISPRi_ASCL1 cells between siASCL1 knockdown NCI-H2107 cells previously profiled by RNA-seq (48) and CRISPRi_KPNB1 NCI-H2107 cell lines. Heatmap shows fold change of each gene with respect to each sample’s control. L and M, GSEA from Ctrl versus CRISPRi_KPNB1 cell lines with normalized enrichment scores (NES) and P values for all genes that were downregulated (L) or upregulated (M) following direct CRISPRi-mediated repression of ASCL1.

Article Snippet: The gene fragment was synthesized (GenScript) and inserted into the TLCV2 backbone in place of Cas9 using AgeI and BamHI sites (TLCV2 was a gift from Adam Karpf; Addgene plasmid #87360; http://n2t.net/ addgene:87360; RRID:Addgene_87360; RRID:Addgene_8736).

Techniques: Targeted Gene Expression, Western Blot, Expressing, Transfection, Control, Comparison, Quantitative RT-PCR, Construct, Knockdown, RNA Sequencing