Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A β-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

doi: 10.1016/j.jcmgh.2021.12.014

Figure Lengend Snippet: Cas9 deletion confirms a super-enhancer necessary for β-catenin-TCF-sensitive locus control. DLD1(DNTCF) cells expressing Cas9 and untargeted (clone #1) or GUCA2A-locus-targeted gRNAs harbor biallelic deletions encompassing the GUCA2A promoter (clone #2), DNase site #6 (clone #3), DNase site #7 (clone #4), or both DNase sites (clone #5). Expression of ( A ) GUCA2A protein, ( B ) GUCA2A mRNA, and ( C ) GUCA2B mRNA was quantified with (+) or without (–) 1 μg/mL DOX for 24 hours. ( D ) Hypothetical model of GUCA2A and GUCA2B promoter positioning relative to a Pol II-rich super-enhancer region upstream of GUCA2A, consisting of multiple DNase sites. ( E ) ChIP-pcr in HT29(APC) cells treated with (+) or without (–) 300 μM zinc for 24 hours reveals enrichment of TCF at the promoter of a Wnt target gene, SP5, with a TCF-specific antibody, but not with control IgG. In contrast, TCF was not detected at sites within 6kb of the GUCA2A TSS: DNase site #5 (GUCA2A promoter), site #6, site #7, or site #8. ( F ) GUCA2A mRNA expression in HT29(APC) cells stably expressing an untargeted (Ctr) or TCF-targeted shRNA, and treated with (+) or without (–) 300 μM zinc for 24 hours. GUCA2A mRNA expression is retained despite TCF knockdown, illustrated by Western blot. ( A–C ) Bars represent the average of ( A ) 2 or ( B , C ) 3 independent experiments ± SEM, and data are presented relative to noninduced cells. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. ( E ) Bars represent the mean ± SD of 3 IPs, and data are presented relative to input DNA. Significance was determined by 2-way analysis of variance. ( F ) Data points represent the average of 3 wells of cells from a single experiment, with the mean of 2 independent experiments indicated. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. Data are presented relative to noninduced cells receiving control shRNA. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Blots were blocked for 1 hour in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 10% milk, then incubated overnight at 4°C with primary antibodies at the indicated dilutions targeting GAPDH (#2118, 1:5000), APC (#2504, 1:1000), β-catenin (#8480, 1:1000), TCF (#2569, 1:1000), Histone H3 (#4499, 1:1000), or Cas9 (#19526, 1:1000) from Cell Signaling Technology (Danvers, MA), or GUCA2A (#HPA018215, 1:250) from Sigma-Aldrich.

Techniques: Expressing, Stable Transfection, shRNA, Western Blot, Transformation Assay

Journal: bioRxiv

Article Title: Guide-specific loss of efficiency and off-target reduction with Cas9 variants

doi: 10.1101/2023.03.16.532856

Figure Lengend Snippet: (A, B) Left: scatter plot showing the categorization of sgRNAs into three groups: “Inefficient” (grey), “Both efficient” (blue), and “WT efficient only” (red), based on the screens with (A) HiFi and (B) LZ3. Right: the log odds ratios of nucleotide frequency between “Both efficient” and “WT efficient only” groups. The data were retrieved by combining all sgRNAs targeting essential genes in the EpiC and Tiling libraries. (C) Left: scatter plot showing the three sgRNA groups defined based on the indel rates in a published dataset using WT SpCas9 and the Cas9 variant HF1 . Right: the log odds ratios of nucleotide frequency between the groups of “Both efficient” and “WT efficient only”. (D) The KL divergence representing the significance of nucleotide difference between the “WT efficient only” and “Both efficient” groups at each position of the spacer. The KL divergence was averaged among the variants HiFi, LZ3, and HF1. (E) Upper: the point mutations (red asterisks) introduced to HiFi, LZ3 and HF1. Bottom: the structural representation of local interactions between the RNA/DNA heteroduplex and REC3 domain of WT SpCas9 (PDB: 7S4U). The positions 9-11 interacting with residues are marked in blue, the positions 15-17 interacting with residues are marked in red, and the residues mutated in any of the three Cas9 variants are marked with asterisks.

Article Snippet: Proteins were separated on 4-15% gradient gels (Bio-Rad) and transferred onto 0.45-μM PVDF membranes, followed by blocking with 5% non-fat milk and incubation with primary antibody against Cas9 (CST, #14697, 1:2,000), YAP1 (CST, #14074, 1:2,000), MTAP (CST, #4158, 1:2,000) or β-actin (Sigma, #A5316, 1:10,000).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Guide-specific loss of efficiency and off-target reduction with Cas9 variants

doi: 10.1101/2023.03.16.532856

Figure Lengend Snippet: (A) Schematic plot of the synthetic dual-target system for the evaluation of off-target effects for a specific sgRNA-target pair. (B) Boxplot comparing the off-on ratios of sgRNA-target pairs harboring 1-, 2-, or 3-mismatch (MM) among the screens with WT SpCas9, HiFi and LZ3. (C) Pairwise comparison of the off-target rates of sgRNA-target pairs among the screens with WT SpCas9, HiFi and LZ3. The consistency of off-target effects is measured in Pearson correlation. (D) Scatter plot comparing the off-on ratios of all the 1-MM sgRNA-target pairs between WT SpCas9 and the Cas9 variants (VT: average off-on ratio of HiFi and LZ3). The sgRNA-target pairs are assigned to three groups based on the off-target effects with WT SpCas9 and the degree of off-target reduction by the variants. (E) Boxplot showing the variant-mediated off-target reduction at each mismatch position measured as the ratio of off-target effects (off-on ratio) between the variants and WT SpCas9 (VT/WT ratio). The p -value is calculated using the two-tailed Manny-Whitney U test. (F) The median VT/WT ratios corresponding to different mismatch contexts between the sgRNA and the target DNA. (G) Comparison of VT/WT ratios at indicated percentile of expected energy barriers caused by the mismatches during R-loop formation. (H) Scatter plot showing the association between the energy barrier caused by a specific mismatch type and the median VT/WT ratio for that mismatch type. rXdY refers to mismatch type where the sgRNA sequence is X and the target DNA sequence is Y.

Article Snippet: Proteins were separated on 4-15% gradient gels (Bio-Rad) and transferred onto 0.45-μM PVDF membranes, followed by blocking with 5% non-fat milk and incubation with primary antibody against Cas9 (CST, #14697, 1:2,000), YAP1 (CST, #14074, 1:2,000), MTAP (CST, #4158, 1:2,000) or β-actin (Sigma, #A5316, 1:10,000).

Techniques: Variant Assay, Two Tailed Test, Sequencing

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Changes of stress granules, endoplasmic reticulum stress and apoptosis levels with the change of hypoxia time. A and B: The level of apoptosis was detected by flow cytometry; C and D: The detection kits were used to evaluate the content of HIF-1α in homogenates, lactate dehydrogenase in cell supernatant; E: The expression of G3BP1 in cells was detected by immunofluorescence; F: The expression of TIA-1 in cells was detected by immunofluorescence; G: The histogram displays the amount of molecules expression; H-J: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 3. Data was shown mean ± SD. a P < 0.05 vs 0 h group; b P < 0.05 vs 4 h group; c P < 0.05 vs 8 h group.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Effects of stress granules agonist on endoplasmic reticulum stress and apoptosis in hepatocyte hypoxia model. A and B: The level of apoptosis was detected by flow cytometry; C and D: The detection kits were used to evaluate the content of HIF-1α in homogenates, lactate dehydrogenase in cell supernatant; E: The expression of G3BP1 in cells was detected by immunofluorescence; F: The expression of TIA-1 in cells was detected by immunofluorescence; G: The histogram displays the amount of molecules expression; H-J: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 3. Data was shown mean ± SD. a P < 0.05 vs normal group; b P < 0.05 vs hypoxia group.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Effect of stress granules on G3BP1 and endoplasmic reticulum stress related molecules in hepatocyte hypoxia model through endoplasmic reticulum stress. A: The expression of G3BP1 in cells was detected by immunofluorescence; B: The expression of TIA-1 in cells was detected by immunofluorescence; C: The histogram displays the amount of molecules expression; D-F: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 3. a P < 0.05 vs normal group; b P < 0.05 vs hypoxia group; c P < 0.05 vs hypoxia + siRNA-ATF4.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Effect of stress granules agonist on G3BP1, HIF-1α, lactate dehydrogenase and endoplasmic reticulum stress related molecules in acute liver failure mice liver. A: The expression of G3BP1 and TIA-1 in liver was detected by immunofluorescence; B: The histogram displays the amount of molecules expression; C and D: The detection kits were used to evaluate the content of HIF-1α in liver tissue homogenate, lactate dehydrogenase in serum; E-G: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 10. Data was shown mean ± SD. a P < 0.05 vs normal group; b P < 0.05 vs model group.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

doi: 10.1101/2023.02.22.529457

Figure Lengend Snippet: (A) Schematic of the CRISPR/Cas9 screening. Stable Cas9 + JJN3 or Cas9 + KMS11 cells were transduced with a library of pooled sgRNAs targeting 196 genes involved in several DNA repair pathways. A portion of cells was collected as a reference sample after 48 hours of transduction. Cells were continuously cultured under puromycin selection and treated with NT or ILF2 ASOs for 3 weeks. ILF2 sensitizer genes were identified using deep sequencing of the sgRNA barcodes and the drugZ algorithm to assess differences in the representation of all sgRNAs between NT ASO– and ILF2 ASO–treated cells across the 3 independent sets of experiments. NGS, next-generation sequencing. (B) Ranking of the DNA repair genes whose sgRNAs were significantly depleted in ILF2 ASO–treated JJN3 cells as compared with NT ASO–treated cells. The inset shows genes on the top ranks (adjusted P <0.01). (C) Western blot analysis of DNA2 in whole-cell lysates (W), nuclei (N), and mitochondria (M) isolated from JJN3 cells. Vinculin, Lamin A, and COX IV were used as the loading controls for W, N, and M, respectively. (D) Kaplan–Meier plots of progression-free survival (PFS) according to DNA2 expression in MM PCs as evaluated by microarray analysis. Shown are the median progression-free survival durations of patients who were enrolled in the Arkansas Total Therapy 2 trial and received high-dose chemotherapy followed by stem cell transplantation (n=256; P= 0.0126). (E) Frequencies of apoptotic JJN3 cells after 3 weeks of exposure to NT or ILF2 ASOs followed by 48 hours of treatment with vehicle (Veh) or NSC at 2 μM. Data are expressed as the mean ± S.D. from one representative experiment performed in triplicate. Statistically significant differences were detected using one-way ANOVA (**** P <0.0001; *** P <0.001). (F) Representative Western blot analysis of ILF2, cleaved caspase 3, and γH2AX in JJN3 cells treated with NT or ILF2 ASOs for 3 weeks prior to receiving NT or ILF2 ASOs alone (Veh) or in combination with 1 μM NSC for 48 hours. Vinculin was used as a loading control. (G) Differences in the luciferase signal in NSG mice engrafted with ILF2 ASO–resistant GFP + Luc + JJN3 cells after receiving NT or ILF2 ASOs alone (NT or ILF2+Veh) or in combination with NSC every day for 7 days. Data are expressed as the mean bioluminescence activity relative to that of the NT ASOs+Veh group [Δ flux of luciferase signal (photons/second, p/s] ± S.D. for each mouse (NT ASOs+Veh, n=22; NT ASOs+NSC, n=15; ILF2 ASOs+Veh, n=19; ILF2 ASOs+NSC, n=11; n=3 independent experiments). Statistically significant differences were detected using one-way ANOVA (** P <0.01; * P <0.05).

Article Snippet: The primary antibodies anti-ILF2/NF45 (Santa Cruz, sc365068), anti-vinculin (Sigma, V9131), anti-γH2AX (Cell Signaling, 2577S), anti-cleaved caspase 3 (Cell Signaling, 966S), and anti-Cas9 (Cell Signaling, 14697S), in addition to secondary anti-mouse and anti-rabbit digital antibodies (Kindle Biosciences LLP), were used.

Techniques: CRISPR, Transduction, Cell Culture, Selection, Sequencing, Next-Generation Sequencing, Western Blot, Isolation, Expressing, Microarray, Transplantation Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

doi: 10.1101/2023.02.22.529457

Figure Lengend Snippet: (A) Western blot analysis of ILF2, γH2AX, cleaved caspase 3, and Cas9 in NT ASO– or ILF2 ASO–treated KMS11 (left) and JJN3 (right) cells after 3 weeks of culture. The 3 biological replicates from the experiment described in are shown (#1-3). Vinculin was used as a loading control. (B) Correlation of the sgRNAs’ gene-level log 2 fold changes in KMS11 (left) and JJN3 (right) cells among the 3 independent sets of experiments. (C) Density functions of gene-level log 2 fold changes (FC) of essential and non-essential genes in KMS11 (left) or JJN3 (right) samples collected after 3 weeks of NT (top) or ILF2 (bottom) ASO treatment. (D) Ranking of DNA repair genes whose sgRNAs were significantly depleted in ILF2 ASO–treated KMS11 cells as compared with NT ASO–treated cells. The inset shows genes on the top ranks (adjusted P <0.01). (E) Representative immunofluorescence images of DNA2 in JJN3 cells. Image was captured and processed using a Delta Vision OMX V4 Blaze Super-Resolution System. Green indicates DNA2; red, TOM20 (mitochondrial marker); and blue, DAPI. Scale bars represent 5 μm. (F) Violin plot of DNA2 expression in the PCs of newly diagnosed MM patients (n=543). Samples were divided into 2 groups (with or without the 1q21 amplification). The lines inside each violin plot define the 4 quartiles of DNA2 expression. Statistically significant differences were detected using a 2-tailed Student t-test (**** P <0.0001). (G) Kaplan–Meier plots of progression-free survival (PFS) according to DNA2 expression in MM PCs. Shown are the median progression-free survival durations of patients who received PIs alone (n=129; left; P= 0.0182); patients who received PIs in combination with other therapies (n=326; middle; P< 0.0001); and patients who received immunomodulatory drugs (n=37; right; P= 0.5682). (H) Frequencies of live JJN3 cells after treatment with NSC at the indicated concentrations for 24, 48, and 72 hours. Data from one representative experiment performed in triplicate are expressed as the mean frequencies ± S.D. of live cells among all cells at each timepoint. (I) Left, frequencies of apoptotic JJN3 cells after 3 weeks of NT ASO or ILF2 ASO exposure followed by 48 hours of treatment with vehicle (Veh) or the DNA2 inhibitor C5 (C5) at the indicated concentrations. Data are expressed as the mean ± S.D. from one representative experiment. Statistically significant differences were detected using one-way ANOVA (**** P < 0.0001; * P <0.05). Right, representative Western blot analysis of ILF2 and cleaved caspase 3 in JJN3 cells treated with NT or ILF2 ASOs alone or in combination with C5 at the indicated concentrations for 48 hours. Vinculin was used as a loading control. (J) Schematic of ASO and NSC treatments in MM xenografts. ILF2 ASO–resistant GFP + Luc + JJN3 cells (1 x 10 6 ) were injected into NSG mice. Ten days after transplantation, mice were injected with luciferin, and tumor burden was quantified using the IVIS Spectrum bioluminescence imaging system. Mice were randomized into 4 groups based on tumor burden on day 0. Mice were injected with NT or ILF2 ASOs alone (25 mg/kg) or in combination with NSC (10 mg/kg) every day for 7 days. Tumor burden was evaluated by bioluminescence imaging on days 0 and 7.

Article Snippet: The primary antibodies anti-ILF2/NF45 (Santa Cruz, sc365068), anti-vinculin (Sigma, V9131), anti-γH2AX (Cell Signaling, 2577S), anti-cleaved caspase 3 (Cell Signaling, 966S), and anti-Cas9 (Cell Signaling, 14697S), in addition to secondary anti-mouse and anti-rabbit digital antibodies (Kindle Biosciences LLP), were used.

Techniques: Western Blot, Immunofluorescence, Marker, Expressing, Amplification, Injection, Transplantation Assay, Imaging

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet: (A) Immunofluorescence staining of THP-1 macrophages in the presence or absence of γ Mtb to identify potential NFAT isoforms of interest in human cells. NFATC2 is most robustly expressed and inducibly nuclear of these isoforms in macrophages. NFATC1 and NFATC3 are comparatively less expressed and less extensively translocated by 8 h post exposure. NFATC4 is very lowly expressed initially but, like all the isoforms, appears to be upregulated at the protein level after γ Mtb exposure. Initial staining from a single experiment, with NFATC2 validation from three biological replicates in (D–G). (B) Magnified images showing robust VEGFA expression in cells with nuclear NFATC2 compared with cells with nuclear NFATC1, NFATC3, and NFATC4, which lack the strong correspondence between NFAT nuclear localization and VEGFA induction, which is only seen with NFATC2 staining. (C) Blinded quantitation of the relationship between each NFAT isoform and the induction of VEGFA. We calculated the subset of cells expressing both VEGFA and demonstrating obvious NFAT nuclear localization and normalized to the total number of cells expressing VEGFA in that field. Initial staining from a single experiment, with validation in (D–G). Total number of cells counted = 5,859. Statistics from ANOVA with Tukey honest significant differences test. (D) THP-1 cells transduced with Cas9-expressing lentiviruses targeting either NFATC2 ( and ) or safe-targeting loci were selected with puromycin and then treated with γ Mtb or vehicle. Safe-targeting-transduced THP-1 cells robustly responded to γ Mtb with VEGFA production at 8 h post exposure, but fewer NFATC2-transduced cells produce VEGFA after stimulation and at lower staining intensity. Representative of three biological replicates. (E) Magnified images showing high VEGFA induction in γ Mtb -treated NFATC2-targeted THP-1 cells compared with γ Mtb -treated safe targeting (ST) THP-1 cells. The ST cells show robust VEGFA induction and NFAT nuclear translocation while NFATC2 cells show diminished VEGFA induction and disordered nuclear translocation and occasional lack of NFATC2 staining entirely; the antibody used to detect NFATC2 is N-terminal to the sgRNA sites, so residual expression is likely captured by the antibody. Representative of three biological replicates. (F) Blinded quantitation of the percentage of VEGFA + cells within entire images for (D and E). Each data point represents the percentage of VEGFA + cells in the field; 15 total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test. (G) Blinded quantitation of the percentage of VEGFA + and nuclear-localized NFAT out of the total number of cells with NFAT nuclear localization within entire images for (D) and (E). Fifteen total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test. (H) Blinded quantitation of the percentage of VEGFA + and nuclear-localized NFAT of the total number of cells in the images for (D) and (E). Fifteen total fields were counted for each group (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test.

Article Snippet: monoclonal mouse anti-Cas9 antibody , Cell Signaling , Cat# 7A9-3A3.

Techniques: Immunofluorescence, Staining, Expressing, Quantitation Assay, Transduction, Translocation Assay

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet:

Article Snippet: monoclonal mouse anti-Cas9 antibody , Cell Signaling , Cat# 7A9-3A3.

Techniques: Recombinant, Cell Culture, Gas Phase Electrophoretic Molecular Mobility Analysis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Software, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Extensive androgen receptor enhancer heterogeneity in primary prostate cancers underlies transcriptional diversity and metastatic potential

doi: 10.1038/s41467-022-35135-2

Figure Lengend Snippet: a Primary PCa tumor AR ChIP-seq log-transformed MACS peak scores for ARBS with H3K27ac HiChIP interaction with CITED2 gene promoter. Gray: no ARBS detected in AR ChIP-seq, Right: Matched log-transformed TMM-normalized RNA-seq expression levels for patients. b Generalized linear model (GLM) -log( p -values) from fitting log-transformed MACS peak scores (predictor) with CITED2 gene expression (response), ARBS were filtered for overlapping H3K27ac presence in LNCaP. Top: Observed model p -value and simulated p -value obtained from permutation tests ( n = 1000) based on likelihood ratio test. c LNCaP single-cell chromatin accessibility for three cell clusters at CITED2 locus with filtered CICERO co-accessibility scores of links for CITED2 enhancers in 80, 54, 10, 8, and 1 patient(s). d Genomic snapshot of CITED2 locus with LNCaP AR ChIP-seq, ranked ARBS from tissue ChIP-seq with CITED2 H3K27ac Hi-ChIP promoter–enhancer pairs found in 80, 54, 10, 8, and 1 patient(s) and design of Cas9 sgRNA pairs (orange, 2 sgRNAs per arrow). e Normalized expression levels of CITED2 over β-actin as measured by RT-qPCR 40 days after infection with non-targeting control (N), sgRNA pair 12 (12), the pool of all sgRNAs guides (p) with gDNA PCR verification of cas9 cut from the same isolate for CITED2 interacting ARBS found in 80, 54, 10, 8, and 1 patient(s). Orange arrows denote cut DNA fragments, nt = nucleotide weight. Representative experiment, center line, mean; error bars, SD; two-tailed Student’s t -test of means on technical replicates, * p < 0.05, ** p < 0.01, *** p < 0.001. f LNCaP proliferation z -score for 878 sgRNAs targeted at AR enhancer locus in Cas9 perturbation or dCas9-KRAB inhibition tiling assay with dotted lines denoting ranked ARBS in 2, 13, 1, and 23 primary tumors. Shaded areas denote 95% confidence interval. g LNCaP proliferation z -score for sgRNAs in ARBS found in 2, 13, 1, and 23 primary tumors, with control comprising z -scores of all other sgRNAs in this region. sgRNAs per ARBS, AR2: 29, AR13: 49, AR23: 49, AR1: 4, ctrl: 1573. Centerline, median; upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Two-tailed Student’s t -test of means with AR23 as reference group, * p < 0.05, ** p < 0.01,*** p < 0.001 **** p < 0.0001, ns non-significant. Source data are provided in Source Data.

Article Snippet: LNCaP cells were infected with a lentivirus encoding Cas9-eGFP (Addgene, 63592) and GFP-positive cells were flow cytometry selected, after which presence of Cas9 in GFP+ cells was confirmed by western blot with antibodies Cas9 (Cell Signaling #14697, clone 7A9-3A3, diluted 1:1000, positive cell line included) and actin (Sigma Aldrich A2228, clone C4, diluted 1:1000).

Techniques: ChIP-sequencing, Transformation Assay, HiChIP, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Infection, Two Tailed Test, Inhibition

Journal: bioRxiv

Article Title: Trem2 Promotes Foamy Macrophage Lipid Uptake and Survival in Atherosclerosis

doi: 10.1101/2022.11.28.518255

Figure Lengend Snippet: A) Trem2 -/- BV2 macrophages were developed using CRISPR/Cas9 deletion approach and differentiated into foamy macrophages by soluble cholesterol treatment. Cells were treated overnight and then assessed for Trem2 expression by flow cytometry. B) WT or (C) Trem2 -/- BV2 macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol overnight, then bulk sequenced by RNA-seq. D) GSEA plot of Cholesterol Biosynthesis pathways. E) Pathway analysis of RNA-seq data comparing WT and Trem2 -/- nonfoamy BV2 cells. E) Pathway analysis of RNA-seq data comparing WT and Trem2 -/- foamy BV2 cells. G) WT or Trem2 -/- BV2 macrophages were differentiated in media control, media with 20 μg/mL or 80 μg/mL soluble cholesterol to induce foamy macrophage formation. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 hours. H) WT or Trem2 -/- BV2 macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then assessed for activation of ER stress response by sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control. (right) Quantification of ER stress response in control or foamy induced WT and Trem2 -/- BV2 macrophages.

Article Snippet: Cas9 expression was validated by flow cytometry using intracellular staining of cas9 protein with anti-cas9 monoclonal antibody (7A9-3A3, Cell Signaling, cat #48796).

Techniques: CRISPR, Expressing, Flow Cytometry, RNA Sequencing Assay, Lactate Dehydrogenase Assay, Activation Assay, Positive Control