spcas9 nickase  (Addgene inc)

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A ) RT-qPCR analysis to monitor efficacy of CRISPR/Cas9-mediated SLFN5 KO in PANC-1 (upper panel) and MIA-Pa-Ca-2 (lower panel) cells. Data are expressed as fold change over WT controls, and bar graphs represent mean ± SEM of 3 independent experiments. **** P < 0.0001. ( B ) Scatter plot (derived from high-throughput single-end RNA-seq analysis) showing the relationship between log 2 (fold change) (LFC) of 347 genes differentially expressed in both SLFN5 -KO ( x axis) and WT ( y axis) PANC-1 cells following IFN-α (5,000 IU for 6 hours) treatment. Select IFN-stimulated genes (ISGs) from the Reactome database ( https://www.reactome.org/content/detail/R-HSA-913531 ; identifier R-HAS-913531) are indicated. The red dashed line deviates from the unity line ( y = x , black line) and represents the linear regression fit ( y = 0.948 x + 0.026), capturing the overall trend between the 2 conditions. The slope ( m = 0.948) indicates a near one-to-one correspondence between the conditions, and the intercept ( c = 0.026) suggests minimal baseline difference. The high coefficient of determination ( R 2 = 0.89) reflects that 89% of the variance in WT LFCs is explained by SLFN5 -KO LFCs. ( C and D ) RT-qPCR analyses of relative mRNA expression of the indicated ISGs in SLFN5 -WT and -KO PANC-1 ( C ) and MIA-Pa-Ca-2 ( D ) cells untreated or treated with human IFN-α (5,000 IU, 6 hours). GAPDH was used for normalization and as an internal control. The data are expressed as fold change over the corresponding untreated cells; bar graphs represent mean ± SEM of 3 ( C ) or 4 ( D ) independent experiments. * P < 0.05. ( E ) PANC-1 SLFN5 -WT and-KO cells were stably transduced with an ISRE-luciferase promoter construct. Cells were incubated for 6 hours in the presence or absence of human IFN-α (5,000 IU) and luciferase activity was measured. Data are expressed as fold increase in luciferase activity in response to IFN-α treatment over control untreated samples for each condition. Bar graphs show mean ± SEM of 3 independent experiments. * P = 0.05. ( F ) ChIP for SLFN5 in PANC-1 cells transduced with lentivirus carrying doxycycline-inducible SLFN5-MYC-FLAG fusion construct. Cells were grown in the presence or absence of doxycycline for 48 hours, followed by IFN-α treatment for 6 hours. qPCR was performed on immunoprecipitated DNA with primers for the ISRE elements in the IFIT1 or ISG15 promoter. Primers for the RPL30 promoter were used as control. Data were normalized to their own IgG control and are expressed as fold enrichment over doxycycline-untreated cells. Shown are mean ± SEM of 3 independent experiments. * P < 0.05. Significance assessed by 2-tailed unpaired t test with Welch’s correction ( A ) or 1-tailed unpaired t test with Mann-Whitney test ( C – F ).

Article Snippet: Briefly, 2 μg of the Cas9 Control Double Nickase plasmid (Santa Cruz Biotechnology, sc437281) or 2 μg of the Slfn5 Double Nickase plasmid (Santa Cruz Biotechnology, sc435875-NIC) were transfected into murine luciferase-expressing KPC1199 cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, CRISPR, Derivative Assay, High Throughput Screening Assay, RNA Sequencing, Expressing, Control, Stable Transfection, Transduction, Luciferase, Construct, Incubation, Activity Assay, Immunoprecipitation, MANN-WHITNEY

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A – C ) RT-qPCR analysis to monitor ( A ) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, ( B ) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and ( C ) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5 -WT and Slfn5 -KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. * P < 0.05, *** P < 0.001 by 2-tailed unpaired t test with Welch’s correction ( A and B ) or 1-tailed unpaired t test with Mann-Whitney test ( C ). ( D ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; ** P < 0.01, **** P < 0.0001. ( E ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; * P ≤ 0.05.

Article Snippet: Briefly, 2 μg of the Cas9 Control Double Nickase plasmid (Santa Cruz Biotechnology, sc437281) or 2 μg of the Slfn5 Double Nickase plasmid (Santa Cruz Biotechnology, sc435875-NIC) were transfected into murine luciferase-expressing KPC1199 cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, CRISPR, Disruption, Expressing, Control, MANN-WHITNEY, Comparison, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: WRN helicase upregulates mitophagy by resolving an intricate nexus of G-quadruplexes-R loops-ATG7 pre-mRNA maturation in cancer

doi: 10.64898/2025.12.11.692796

Figure Lengend Snippet: (A) WRN depleted cells (WRN -/- ) were generated by CRISPR-Cas9 double nickase plasmid system and then the expression was assessed by Western blotting. The blot was probed for WRN and β-actin as a loading control. (B, C) Mitochondrial respiration was measured in WRN +/+ and WRN -/- cells using the Seahorse XF Analyzer. The oxygen consumption rate (OCR) was monitored over time, and phases of basal respiration, proton leak, maximal respiratory capacity and reserve capacity were indicated by grey, orange, green and blue shades, respectively in B. (D, E) NBD-glucose uptake was analyzed by flow cytometry in WRN +/+ and WRN -/- cells, measuring the green fluorescence of NBD-glucose uptake. Relative NBD-glucose uptake was quantified to assess glucose transport in WRN +/+ and WRN -/- cells. (F) Mitochondrial membrane potential was evaluated by flow cytometry using JC-1 dye. The fluorescence emission at red and green channels was analyzed to assess membrane potential in WRN +/+ and WRN -/- cells. (G, H) Mitochondrial morphology was visualized using Mito-Tracker Green staining and quantified the intensity in WRN +/+ and WRN -/- cells. Representative images of mitochondrial fluorescence are shown. (I-K) Mitochondrial morphology and branching were assessed using Mito-Tracker Green staining followed by skeletonizing of mitochondrial structures in WRN +/+ and WRN -/- cells. Representative images are shown. Mitochondrial branching per mitochondrion and total mitochondrial branch length was quantified in WRN +/+ and WRN -/- cells, measuring extent of mitochondrial network. All the values indicated are mean ± SEM (n = 4) ** p < 0.01, * p <0.05.

Article Snippet: Antibodies against p62, WRN (#SC5629) and CRISPR-Cas9 double nickase plasmids (control and WRN) were from Santa Cruz biotechnology (Santa Cruz, CA).

Techniques: Generated, CRISPR, Plasmid Preparation, Expressing, Western Blot, Control, Flow Cytometry, Fluorescence, Membrane, Staining