Journal: bioRxiv

Article Title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

doi: 10.1101/2022.01.20.477122

Figure Lengend Snippet: a. CRISPR/Cas9 DNA editing tool was used to generate deletions (green box) in the highly conserved DBD from vxs1 (top) and vsx2 (bottom) TFs. Blue boxes represent gene exons, black boxes the location of sgRNAs used to guide Cas9 endonuclease and primers for screening are depicted as opposing arrowheads. b-d and f-h. Histological sections stained with nuclear marker DAPI and phalloidin-Alexa488 for actin filaments from WT (b-d, n≥8) and vsx KO central retinas (f-h, n≥10) at 48hpf (b, f), 72hpf (c, g) and 6dpf (d, h). e, i. Head dorsal view from 6dpf WT (e) and vsx KO (i) larvae with insets showing their pigmentation pattern (white arrowhead). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar in b-d and f-h: 50μm, scale bar in e and i: 500μm.

Article Snippet: To target individual vsx genes, a solution containing two sgRNAs (40 ng/μL each) and Cas9 protein (250 ng/μL) (Addgene; 47327) ( ) were injected into one-cell-stage zebrafish and medaka embryos.

Techniques: CRISPR, Staining, Marker

Journal: bioRxiv

Article Title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

doi: 10.1101/2022.01.20.477122

Figure Lengend Snippet: a. CRISPR/Cas9 was used to eliminate (green box) the DBD from vxs1 (top) and vsx2.1 (bottom) TFs in medaka. Blue boxes represent exons, black boxes the location of sgRNAs used and primers for screening are depicted as opposing arrowheads. b-e. Histological sections from WT (b, d, n=4) and vsx KO central retinas (c, e, n=5) at 12dpf. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar b-c: 50μm, d-e: 20 μm.

Article Snippet: To target individual vsx genes, a solution containing two sgRNAs (40 ng/μL each) and Cas9 protein (250 ng/μL) (Addgene; 47327) ( ) were injected into one-cell-stage zebrafish and medaka embryos.

Techniques: CRISPR

Journal: bioRxiv

Article Title: Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

doi: 10.1101/2020.04.30.071571

Figure Lengend Snippet: A. Schematic diagram of CRISPRi system in Enterococcus faecalis. A two-plasmid system consisting of a small (3,182 kb) vector, pGCP123, for sgRNA expression and a 12,621kb plasmid, pMSP3545-dCas9 Str , for dCas9 Str expression. The sgRNA and dCas9 Str are expressed from a nisin-inducible promoter nisA that is activated upon addition of nisin to the media through the NisKR two-component system encoded on pMSP3545-dCas9 Str . The assembled sgRNA-dCas9 complex blocks gene transcription by binding to DNA and blocking RNA polymerase. Image created with BioRender.com. B. Western blot with anti-Cas9 Str antibody on induced EF dCas9 pMSP3545 (empty vector) and EF dCas9 pMSP3545-dCas9 Str at nisin concentration 0-500 ng/ml.

Article Snippet: Cas9 was detected using a monoclonal mouse anti-Cas9 antibody (Abcam).

Techniques: Plasmid Preparation, Expressing, Binding Assay, Blocking Assay, Western Blot, Concentration Assay

Journal: Diabetes & Metabolism Journal

Article Title: N 6 -Methyladenosine Methyltransferase METTL3 Alleviates Diabetes-Induced Testicular Damage through Modulating TUG1/Clusterin Axis

doi: 10.4093/dmj.2021.0306

Figure Lengend Snippet: Effects of methyltransferase like-3 (METTL3) overexpression on the viability and apoptosis of GC-1 spg cells induced by high glucose. Mouse GC-1 spg cells were transfected with or without vectors overexpressing METTL3 or empty control vectors (pcDNA3.1), then cells were cultured in high glucose (HG) medium (30 mmol/L) for 24 hours. The expression of METTL3 in GC-1 spg cells was measured by (A) real-time quantitative polymerase chain reaction and (B) Western blot. (C) Cell viability was evaluated by the MTT assay. (D-H) The levels of malondialdehyde (MDA) and glutathione (GSH) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in GC-1 spg cells treated with or without HG were measured by commercial kits. (H) The level of reactive oxygen species (ROS) was detected using the 2ʹ,7ʹ-dichlorodihydro-fluorescin diacetate (DCFH-AD) probe. (I) Cell apoptosis was determined by flow cytometry. (J) The expressions of Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 were quantified by Western blot. NG, normal glucose. a P <0.05, b P <0.01, c P <0.001.

Article Snippet: After blocking, PVDF membrane was incubated with specific antibodies overnight, including METTL3 (cat# ab195352, Abcam), Bcl-2 (cat# ab32124, Abcam), Bax (cat# ab3191, Abcam), cleaved caspase-3 (cat# ab32499, Abcam), cleaved caspase-9 (cat# ab32539, Abcam), and clusterin (cat# ab269342, Abcam) at 4°C.

Techniques: Over Expression, Transfection, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Flow Cytometry

Journal: Diabetes & Metabolism Journal

Article Title: N 6 -Methyladenosine Methyltransferase METTL3 Alleviates Diabetes-Induced Testicular Damage through Modulating TUG1/Clusterin Axis

doi: 10.4093/dmj.2021.0306

Figure Lengend Snippet: Methyltransferase like-3 (METTL3) regulated the viability and apoptosis of high glucose (HG)-induced GC-1 spg cells via taurine up-regulated 1 (TUG1). GC-1 spg cells were co-transfected with pcDNA3.1-METTL3 vector and si TUG1 for 48 hours. Then, cells were exposed to HG conditions for 24 hours. (A, B) The RNA expressions of TUG1 and clustern were verified by real-time quantitative polymerase chain reaction. (C) Cell viability was evaluated by the MTT assay. (D, E, F, G) The levels of malondialdehyde (MDA) and glutathione (GSH) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in GC-1 spg cells treated with or without HG were measured by commercial kits. (H) The level of reactive oxygen species (ROS) was detected using the 2ʹ,7ʹ-dichlorodihydro-fluorescin diacetate (DCFH-AD) probe. (I) Cell apoptosis was assessed by flow cytometry. (J) The expressions of Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 were quantified by Western blot. NG, normal glucose. a P <0.05, b P <0.01, c P <0.001.

Article Snippet: After blocking, PVDF membrane was incubated with specific antibodies overnight, including METTL3 (cat# ab195352, Abcam), Bcl-2 (cat# ab32124, Abcam), Bax (cat# ab3191, Abcam), cleaved caspase-3 (cat# ab32499, Abcam), cleaved caspase-9 (cat# ab32539, Abcam), and clusterin (cat# ab269342, Abcam) at 4°C.

Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Western Blot

Journal: Diabetes & Metabolism Journal

Article Title: N 6 -Methyladenosine Methyltransferase METTL3 Alleviates Diabetes-Induced Testicular Damage through Modulating TUG1/Clusterin Axis

doi: 10.4093/dmj.2021.0306

Figure Lengend Snippet: Methyltransferase like-3 (METTL3) alleviated diabetes-induced testicular damage in vivo . The diabetic mice were then divided into three groups ( n =10 per group): streptozocin (STZ), STZ+pcDNA3.1, and STZ+METTL3. After treatment, the testicular tissues and blood samples were collected for further detection. (A) The blood glucose level of STZ-treated mice and control mice was measured using a glucometer. (B) The serum level of testosterone was determined by enzyme-linked immunosorbent assay (ELISA). (C) H&E staining was performed to show pathophysiological changes in the testicles. (D) The expression of METTL3 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). (D, E, F, G, H) The levels of malondialdehyde (MDA) and glutathione (GSH) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in mouse testicular tissues were measured by commercial kits. (I) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to determine the apoptosis of cells in testicular tissues. (J) The expressions of Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 were quantified by Western blot. (K) The RNA expression of taurine up-regulated 1 (TUG1) was assessed by RT-qPCR. (L, M) The protein expression of clusterin was measured by immunohistochemistry (IHC) and Western blot. a P <0.05, b P <0.01, c P <0.001.

Article Snippet: After blocking, PVDF membrane was incubated with specific antibodies overnight, including METTL3 (cat# ab195352, Abcam), Bcl-2 (cat# ab32124, Abcam), Bax (cat# ab3191, Abcam), cleaved caspase-3 (cat# ab32499, Abcam), cleaved caspase-9 (cat# ab32539, Abcam), and clusterin (cat# ab269342, Abcam) at 4°C.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, TUNEL Assay, Western Blot, RNA Expression, Immunohistochemistry

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Up-regulated Hallmark and Gene Ontology (GO) pathways in ID8 cell single-guide SMARCA4 (sg SMARCA4 ) compared to those in sgNTC. Ribodeplete RNA sequencing was performed. Statistical analysis was based on hypergeometric test and performed using ClusterProfiler. IL-6, interleukin-6; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; TNFA, tumor necrosis factor–α; FDR, false discovery rate. ( B ) Gene expression heatmap of type I IFN pathway–related genes in ID8 cells. Reads per kilobase of transcript per million mapped reads values were scaled to z -score for visualization. Gene expression fold change of sg SMARCA4 versus sgNTC cells is color coded according to the legend. ( C ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation results for IFN genes in ID8 cells (sgNTC and four sg SMARCA4 clones). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control (sgNTC). n = 3 independent experiments. ( D ) MHC1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. ( E ) PD-L1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. MFI, median fluorescence intensity. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; n.s., not significant. Error bars represent ± SEM. Samples in duplicates [(A) and (B)] and triplicates [(D) and (E)]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Clone Assay, Expressing, Control, Flow Cytometry, Fluorescence, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Changes in genomic site accessibility in sg SMARCA4 (KO1) versus sgNTC cells. Log 2 fold change and FDR-adjusted P value (Wald test P values from DESeq2 with Benjamini-Hochberg correction). ( B ) Gene set enrichment analysis (GSEA) of immune pathways with increased accessibility in sg SMARCA4 versus sgNTC cells. Kolmogorov-Smirnov statistic with Benjamini-Hochberg correction. Exact q values indicated in each panel. ( C ) Changes in chromatin accessibility at transcription start sites (TSSs) of ISGs (CXCL10 and CCL2) in sg SMARCA4 versus sgNTC cells. ( D ) Motifs enriched in open chromosomal regions affected by SMARCA4 deficiency. P value and binomial test were performed using Homer2. Experiments performed in duplicates. GO, Gene Ontology; KO, knockout; KO_1, target exon 14 clone 1; NTC, non-target control; TF, transcription factor.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Knock-Out, Control

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) IFNAR-1 neutralizing assay in ID8 single-guide NTC (sgNTC) compared to that in sg SMARCA4 cells (four sg SMARCA4 clones). Cells were treated with Mock or IFNAR-1 antibody for 48 hours at 10 μg/ml. ( B ) IRF3 expression levels in sg SMARCA4 versus sgNTC cells by qRT-PCR. ( C ) IRF3 expression in doxycycline-inducible short hairpin NTC (shNTC) and shIRF3-transfected sg SMARCA4 cells by qRT-PCR. ( D ) qRT-PCR quantification of ISGs in ID8 sgNTC and sg SMARCA4 cells. The latter were transfected with shIRF3 or shNTC (with and without POLYI:C). ( E ) Enriched IRF motif at TSSs of ISG locus from ATAC-seq analysis of ID8 sgNTC versus sg SMARCA4 cells. Experiments performed in duplicates. ( F ) Analysis of publicly available ChIP-Atlas data of IRF3 DNA binding sites on ISGs in murine immune cell lines. Statistical analysis was performed using two-tailored unpaired t test [(A) to (D)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Error bars represent ± SEM. n = 3 independent experiments in (A) to (D). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sgNTC_1 in (A), (B), and (D) and sg SMARCA4 transfected with shNTC in (C), as indicated]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; LPS, lipopolysaccharide stimulated, NTC, non-target control; NTC_1 = NTC clone 1; POLYI:C, polyinosinic-polycytidylic acid stimulated.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Neutralizing Assay, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Control, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Volcano plots for differential expression of TEs in ID8 cells. ( B ) Heatmap of long terminal repeats (LTR) with adjusted P < 0.05. ( C ) Representative pictures (top) of double-stranded RNA (dsRNA) identification by immunofluorescence (IF) in sg SMARCA4 and sgNTC ID8 cells with quantification (bottom). RNAse, ribonuclease. ( D ) qRT-PCR quantification of ISGs in ID8 sgNTC cells and sg SMARCA4 cells transfected with shMAVS or shNTC. ( E ) MAVS expression in doxycycline-inducible shNTC and shMAVS-transfected sg SMARCA4 cells by qRT-PCR. Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sg SMARCA4 transfected with shNTC in (C) and sgNTC_1 in (D), as indicated]. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Error bars represent ± SEM. Samples in duplicates [for (A) and (B)]. n = 3 independent experiments [in (C) and (D)]. Red line in (A) represents a cutoff of an adjusted P value of <0.05. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Immunofluorescence, Quantitative RT-PCR, Transfection, Expressing, Control, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Workflow for the OC tumor model. C57BL/6 (Cg)–Tyr c-2J /J mice were inoculated intraperitoneally with 10 million ID8 sgNTC or sg SMARCA4 ( KO_4 ) tumor cells, and spectral flow cytometry was performed 21 days later on harvested ascites samples. ip, intraperitoneal. ( B ) Frequency of tumor PD1 + CD4 + T cells and PD1 + CD8 + T cells. ( C ) Frequency of NK1.1 + cells expressing Granzyme B + . ( D ) Frequency of tumor dendritic cells (of CD45 + cells) and MHCII-expressing dendritic cells. ( E ) Frequency of tumor macrophages (of CD45 + cells) and PD-L1–expressing macrophages. ( F ) In vivo bioluminescence imaging of tumor burden in sg SMARCA4 versus sgNTC tumors (unpaired t test of area under the curve). Statistical analysis was performed using two-tailored unpaired t test [(B) to (F)]. * P < 0.05, ** P < 0.01 and **** P < 0.0001. Error bars represent ± SEM. n = 10 mice per group. KO, knockout; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, In Vivo, Imaging, Knock-Out, Control

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Workflow for the B16-F10 tumor model. C57BL/6 (Cg)–Tyr c-2J /J mice were inoculated subcutaneously with 150,000 shNTC, sgEV, sh SMARCA4_1 , sh SMARCA4_2 , or sg SMARCA4 tumor cells, and spectral flow cytometry was performed 21 days later on harvested tumor samples. ( B ) Frequency of tumor CD8 + T cells and MFI of ICOS + -expressing CD8 + T cells in B16-F10 model. ( C ) Frequency of NK1.1 + cells in B16-F10 knockout and knockdown models (of CD45 + cells). ( D ) MFI of MHCII in macrophages in B16-F10 SMARCA4 knockout and knockdown models. ( E ) Tumor volume of sh SMARCA4 versus shNTC B16 tumors (unpaired t test of final time points). n = 5 mice per group, three biological replicates. ( F ) Frequency of NK1.1 + cells in sh SMARCA4 versus shNTC tumors (of live cells) in RAG2 −/− mice. ( G ) MFI of MHCII-expressing macrophages in B16-F10 sh SMARCA4 versus shNTC tumors in RAG2 −/− mice. ( H ) Tumor volumes of sh SMARCA4 versus shNTC B16 tumors in RAG2 −/− mice (unpaired t test of final time points). n = 3 to 5 mice per group, three biological replicates. Statistical analysis was performed using two-tailored unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent ± SEM. Dox, doxycycline; EV, empty vector; NTC, non-target control; SC, subcutaneous.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Knock-Out, Knockdown, Plasmid Preparation, Control