lentiv cas9 blast  (Addgene inc)

Journal: Nature Communications

Article Title: KLF5 enables dichotomous lineage programs in pancreatic cancer via the AAA+ ATPase coactivators RUVBL1 and RUVBL2

doi: 10.1038/s41467-025-66007-0

Figure Lengend Snippet: A Western blot of KLF5 in AsPC-1 and T3M-4 in which endogenous KLF5 is replaced with 3xFLAG-FKBP12 F36V -KLF5. Whole-cell lysates collected following 300 nM dTAGv-1 treatment for indicated times. Representative of 3 biological replicates. β-actin, loading control. B – D SLAM-seq following 4-h total treatment with 300 nM dTAG v -1 or dTAG v -1-NEG (control), including 2-h 4sU labeling. Representative of 2 (T3M-4) or 3 (AsPC-1) biological replicates. Fold change and significance calculated by DESeq2 . B Gene set enrichment analysis of differentially expressed transcripts following dTAG v -1. Significance by GSEA. NES normalized enrichment score, FWER family-wise error rate. C , D Volcano plots of differentially expressed transcripts following dTAG v -1. Select Classical and Basal-like genes (Moffitt et al. ) and SERPINB5 labeled. E – K KLF5 ChIP-seq in two classical (AsPC-1 and HPAF-II) and two basal-like (BxPC-3 and T3M-4) lines. Classical, Basal-like, and Common sets defined by shared peaks (MACS2 q < 0.01, bedtools intersect). n = 2 independent IPs per line. E Heatmap of 4394 KLF5 peaks. Rows = 10Kb genomic regions centered on a KLF5 peak, ordered by KLF5 signal. F , G Representative KLF5 and H3K27ac ChIP tracks, visualized in UCSC genome browser . Matched, scaled track heights indicated (right). H , I H3K27ac ChIP-seq following 3.5-h dTAG v -1 or DMSO (control) treatment. Metagene plots show average H3K27ac signal at all KLF5 peaks in each peak set. J , K HOMER motif enrichment for subtype-specific KLF5 peaks. The top 4 transcription factor family motifs were selected. L Competition-based fitness assays in Cas9-expressing SUIT-2, AsPC-1, T3M-4, and BxPC-3 cells after CRISPR-Cas9 knockout (KO) of KLF5 or ROSA26 (control) using sgRNAs coupled to GFP. Bars = mean ± SD of normalized %GFP (to day 3 post-infection). Two independent sgRNAs per KO. n = 3 (SUIT-2, T3M-4) or 5 (AsPC-1, BxPC-3) biological replicates. Significance reported in Source data. L , N , O Significance assessed by Generalized Additive Mixed Model. M Schematic of orthotopic transplantation of luciferase+ human PDAC cell lines. Created in BioRender. Cunniff, P. (2025) https://BioRender.com/8362080 . N – P n = 5–6 mice per group. N , O Bioluminescence of orthotopic PDAC tumors. Lighter lines: individual mice. Dark line: mean(log 10 (luminescence)). P Western blot of KLF5 after KLF5 or ROSA26 (control) KO (day 5) or from freshly resected primary tumors. β-actin, loading control. Representative of two Western blot replicates. Source data are provided as a Source Data file.

Article Snippet: For GFP-depletion assays, cells stably expressing Cas9 from a LentiV-Cas9-blasticidin vector were lentivirally transduced with sgRNAs in an LRG2.1_puromycin vector (Addgene # 125594) at a ~30–50% GFP-positivity rate.

Techniques: Western Blot, Control, Labeling, ChIP-sequencing, Expressing, CRISPR, Knock-Out, Infection, Transplantation Assay, Luciferase

Journal: Nature Communications

Article Title: KLF5 enables dichotomous lineage programs in pancreatic cancer via the AAA+ ATPase coactivators RUVBL1 and RUVBL2

doi: 10.1038/s41467-025-66007-0

Figure Lengend Snippet: A ChIP-seq tracks of KLF5 and H3K27ac enrichment at the SERPINB5 locus, visualized in UCSC genome browser . Matched, scaled track heights indicated (right). B Flow cytometry analysis of AsPC-1 and T3M-4 cells, methanol-fixed and stained with anti-Serpin B5 antibodies 7 days following CRISPR-Cas9 knockout (KO) of SERPINB5 , KLF5 , or control ( ROSA26 ). Representative of 4 independent experiments. Gating shown in Supplementary Fig. H. C Workflow of genome-wide Serpin B5 reporter screen. Created in BioRender. Cunniff, P. (2025) https://BioRender.com/l7na7wx . Corepressors and coactivators refer to cofactors that repress or promote KLF5 transcriptional activity, respectively. D Genome-wide Serpin B5 reporter screen results in AsPC-1. Genes (dots) ordered alphabetically along the x-axis. MBP-KLF5 IP Mass Spectrometry interactors from AsPC-1 nuclear lysate labeled with purple dots. Select outlier genes labeled. Beta scores and significance calculated using MAGeCK (maximum likelihood estimation). Negative beta scores indicate enrichment in the Serpin B5 low population. Gating shown in Supplementary Fig. I. E Log 10 -transformed label-free quantification (LFQ) of peptides mapping to the indicated proteins by Mass Spectrometry. MBP alone is a negative control. n = 3 independent IPs. Significance assessed by unpaired two-tailed Student’s t -test (reported in Source Data). F Western blot of KLF5 and Serpin B5. Protein lysates were collected from AsPC-1 and T3M-4 on day 7 following KO of the indicated genes or ROSA26 (control). Representative of 2 (T3M-4) or 3 (AsPC-1) biological replicates. β-actin, loading control. G , H RNA-sequencing performed on day 5 following KO of the indicated genes or ROSA26 (control). 2 independent sgRNAs were used for KLF5 , RUVBL1 , RUVBL2 , and ROSA26 . n = 2 per sgRNA. Fold change and significance of differentially expressed genes (DEGs) following KO by DESeq2. G Gene set enrichment analysis of the DEGs following RUVBL1 KO. Significance calculated by GSEA. NES normalized enrichment score, FWER family-wise error rate. H Heatmap of z-scores of variance-stabilized normalized gene counts for the top 1000 DEGs following KLF5 knockout. Columns = samples. Rows = genes. Samples were clustered using Euclidean distance (dendrogram, top). Variance stabilized transformed counts calculated with DESeq2. Select genes associated with basal-like PDAC (Moffitt et al. ) labeled (right). Source data are provided as a Source Data file.

Article Snippet: For GFP-depletion assays, cells stably expressing Cas9 from a LentiV-Cas9-blasticidin vector were lentivirally transduced with sgRNAs in an LRG2.1_puromycin vector (Addgene # 125594) at a ~30–50% GFP-positivity rate.

Techniques: ChIP-sequencing, Flow Cytometry, Staining, CRISPR, Knock-Out, Control, Genome Wide, Activity Assay, Mass Spectrometry, Labeling, Transformation Assay, Quantitative Proteomics, Negative Control, Two Tailed Test, Western Blot, RNA Sequencing