Journal: Nature Communications

Article Title: CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

doi: 10.1038/s41467-020-18936-1

Figure Lengend Snippet: The JEV-infection cycle starts with binding to co-factors HSPGs, and/or unknown cellular receptors, followed by viral entry to enable replication. The EMC complex protein (EMC3 and EMC6) and CALR calcium-binding protein of the ER lumen are involved in JEV replication of host cells. Subsequently, the JEV RNA genome is replicated, viral particles are matured and packaged, and are released from cells. JEV, Japanese encephalitis virus; HSPG, heparan sulfate proteoglycan; PAPS, 3′-Phosphoadenosine-5′-phosphosulfate; PAP, 3′-phosphoadenosine-5′-phosphate; ATP, Adenosine triphosphate; ADP, Adenosine diphosphate; APS, Adenosine 5′ phosphosulfate; PPI, pyrophosphate; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein degradation; EMC, endoplasmic reticulum membrane protein complex.

Article Snippet: Separated proteins were then transferred onto a nitrocellulose membrane and probed with Cas9 (GeneTex, #GTX53807, 1:3000), EMC3 (Santa Cruz Biotechnology, #sc-365903, 1:500), CALR (Abclonal, #A1066, 1:1000) antibody, with β-tubulin (Sungenebiotech, #KM9003T, 1:5000)/GAPDH antibodies (Beyotime, #AF5009, 1:3000) used as an internal loading control.

Techniques: Infection, Binding Assay, Virus, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome analysis reveals determinant stages controlling human embryonic stem cell commitment to neuronal cells

doi: 10.1074/jbc.M117.796383

Figure Lengend Snippet: Generation of SIX3 and HESX1 knock-down hESC lines using CRISPR/Cas9 system. A, Western blot for CRISPR/Cas9 efficiency validation of SIX3 and HESX1 knockdown at day 12 differentiation samples. SIX3 is about 37 kDa; HESX1 is about 47 kDa, and GAPDH is 34 kDa. B, RT-qPCR analysis for the gene expression at day 12 in the SIX3 knock-out and HESX1 knockdown cell lines. C, Western blot for CRISPR/Cas9 efficiency validation of PAX6 knockdown at day 12 differentiation samples. PAX6 is about 50 kDa. D, double immunocytochemistry analysis of OCT4 and SOX2, OTX2 and SOX2 respectively, on day 6. Scale bars, 75 μm. E, quantification of the immunocytochemistry in D. F, immunocytochemistry analysis of PAX6 on day 12. Scale bars, 75 μm. G, quantification of the immunocytochemistry in F. H, immunocytochemistry analysis of NEUN on day 50. Scale bars, 75 μm. I, quantification of the immunocytochemistry in H. To quantify the differentiation efficiency, three to five fields were randomly selected. n = 3 independent experiments; two-tailed t test. All data are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01. Related to E, G, and I.

Article Snippet: The primary antibodies are SIX3 (Abcam, catalog no. {"type":"entrez-nucleotide","attrs":{"text":"AB172131","term_id":"90079026","term_text":"AB172131"}} AB172131 ), HESX1 (ABclonal, catalog no. {"type":"entrez-nucleotide","attrs":{"text":"A10696","term_id":"490814","term_text":"A10696"}} A10696 ), PAX6 (Covance, catalog no. AB2237), and GAPDH (ABclonal, catalog no. AC002) ( supplemental Table S3 ).

Techniques: CRISPR, Western Blot, Quantitative RT-PCR, Expressing, Knock-Out, Immunocytochemistry, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome analysis reveals determinant stages controlling human embryonic stem cell commitment to neuronal cells

doi: 10.1074/jbc.M117.796383

Figure Lengend Snippet: qPCR analysis of downstream target gene of SIX3 and HESX1 in CRISPR/Cas9 knock-out cell lines. A, regulation network of hub TF SIX3. B, relative expression level of SIX3 downstream target genes in KO cell lines at day 0. C, relative expression level of SIX3 downstream target genes in KO cell lines at day 22. D, regulation network of hub TF HESX1. E, relative expression level of HESX1 downstream target genes in KD cell lines at day 6. F, relative expression level of HESX1 downstream target genes in KD cell lines at day 22. For q-PCR, a minimum of three biological replicates from two separate experiments were examined.

Article Snippet: The primary antibodies are SIX3 (Abcam, catalog no. {"type":"entrez-nucleotide","attrs":{"text":"AB172131","term_id":"90079026","term_text":"AB172131"}} AB172131 ), HESX1 (ABclonal, catalog no. {"type":"entrez-nucleotide","attrs":{"text":"A10696","term_id":"490814","term_text":"A10696"}} A10696 ), PAX6 (Covance, catalog no. AB2237), and GAPDH (ABclonal, catalog no. AC002) ( supplemental Table S3 ).

Techniques: CRISPR, Knock-Out, Expressing

Journal: Therapeutic Advances in Chronic Disease

Article Title: N 6 -methyladenosine demethylases Alkbh5/Fto regulate cerebral ischemia-reperfusion injury

doi: 10.1177/2040622320916024

Figure Lengend Snippet: Betaine treatment enhanced OGD/R treated primary neuronal death and CL treatment attenuated OGD/R-treated primary neuronal death. (A–C) Primary neurons were treated with 8 mM Betaine or 20mM CL and then cultured under OGD/R conditions or normoxia. Cell death was quantified by CCK8 assay or by microscopy for PI-positive cells. Quantifications of the percentage of dead cells were shown. (mean ± SEM; n = 6–20; * p < 0.05, *** p < 0.001 versus OGD/R). (D–E) Representative images of Western blots of primary neuron treated with 8 mM Betaine or 20 mM CL. Cleaved-caspase3 levels were determined by Western blot (mean ± SEM; n = 3; * p < 0.05 versus OGD/R). (F) A proposed model for Alkbh5 and Fto in cerebral ischemia-reperfusion injury. CCK8, Cell Counting Kit-8; CL, cycloleucine; OGD/R, glucose oxygen deprivation/reoxygenation; PI, propidium iodide; SEM, standard error of the mean.

Article Snippet: Neurons were cultured in vitro for neuron standard medium supplemented with cycloleucine (CL) [A1063, TCI (Shanghai) Chemical Industry Development Co., Ltd.] (10 mM, 20 mM, 40 mM) and betaine (61962, Sigma) (4 mM, 8 mM, 16 mM) from 24 h before OGD to the end of the experiment.

Techniques: Cell Culture, CCK-8 Assay, Microscopy, Western Blot, Cell Counting

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: GCH1 was upregulated in niraparib-treated groups. (a) Heatmap and (b) volcano plots of differentially expressed metabolism-related genes under niraparib administration.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques:

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: GCH1 was increased upon niraparib treatment at transcriptional and translational levels. GCH1 transcription was analyzed by (a) qRT-PCR in MDA-MB-231, A2780 and HCC38 cell lines after treated with 10 μ M niraparib for 48 h. The expression of GCH1 was measured by (b) western blot in MDA-MB-231, A2780 and HCC38 cell lines after treated with 10 μ M niraparib for 72 h. (c) Representative immunofluorescence images of GCH1 expression in control group and cells treated with 10 μ M niraparib for 72 h. (d) Representative images of IHC staining of GCH1 in human ovarian and breast cancer tissues derived from PDX models are shown, and GCH1 expression was quantified according to the IHC score.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Control, Immunohistochemistry, Derivative Assay

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: Association between GCH1 expression and the clinicopathological characteristics. GCH1 expression is higher in (a) ovarian tumor and (b) breast tumor in the TCGA and GTEx database. (c) Increased expression of GCH1 in breast cancerous tissue compared with paired normal tissue. GCH1 is correlated with (d) histological type, (e) PR status, (f) ER status, (g) HER2 status, and (h) PAM50 subtype classification in BRCA. GCH1 was not significantly correlated with (i) pathologic stage, (j) N stage, (k) M stage, and (l) T stage in breast cancer cohort. (m) Histologic stage, (n) lymphatic invasion, and (o) venous invasion were not significantly correlated with GCH1 expression in ovarian cancer cohort.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques: Expressing

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: Identification of GCH1-associated DEGs and the potential transcription factor STAT1 of GCH1. Heat map showing the top 50 DEGs between high- and low-GCH1 expression groups in (a) BRCA and (b) OV. GSEA showing the enrichment of the JAK-STAT pathway in GCH1-high expression (c) breast and (d) ovarian cancer. Shown are correlation of GCH1 and STAT1 expressions in (e) BRCA and (f) OV cohorts, respectively. (g) Cistrome Data Browser indicated STAT1 regulated GCH1 transcription directly. (h) GCH1 mRNA were evaluated by qRT-PCR in MDA-MB-231, A2780, and HCC38 cells when treated with 10 μ M niraparib and/or 50 μ M JAK inhibitor for 48 h. (i–k) The protein level of GCH1 were determined by Western blot in MDA-MB-231, A2780, and HCC38 cells after treated with 10 μ M niraparib and/or 50 μ M JAK inhibitor for 72 h.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: GCH1 was associated with homologous recombination repair (HRR) pathway. GSEA analysis of differentially expressed genes between high- and low-GCH1 expression groups showing the enrichment of homologous recombination repair (HRR) pathway in (a) breast cancer and (b) ovarian cancer. (c-d) Shown are expression profiles of 28 HRR-related genes in high- and low-GCH1 groups, and data are presented by heatmaps. Correlation diagrams showing the association between GCH1 expression and HRR-related genes, including FANCA and BRCA2 in (e) breast cancer and RAD54L and FANCI in (f) ovarian cancer. Representative immunofluorescence images and quantification of γ -H2AX foci and Rad51 foci formation in control and 5 mM DAHP-treated groups of (g, i) MDA-MB-231 cells and (h, j) A2780 cells. 10 μ M niraparib was used as positive control and all the drugs were administered for 24 hours.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques: Homologous Recombination, Expressing, Immunofluorescence, Control, Positive Control

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: Inhibition of GCH1 potentiated PARP inhibitor therapy for cancers. (a) Quantitative real-time PCR assay of GCH1 in MDA-MB-231 cells 48 hours after transfection with indicated siRNAs. (b) Apoptosis of MDA-MB-231 cells was measured via flow cytometry after 48 hours of siRNA transfection. (c) MDA-MB-231 cells were treated with 10 μ M niraparib for indicated times after transfection with siRNAs and assessed by flow cytometry for annexin V-FITC and propidium iodide (PI) staining. Plots are representative of three independent experiments and the percentages of annexin V positive cells are quantified. (d) MDA-MB-231 cells viability in presence of increasing amounts of DAHP. Nonlinear fitting showed the corresponding IC50. (e) MDA-MB-231 cells and (f) A2780 cells were treated with control, 5 mM DAHP, 10 μ M niraparib, or a combination for 72 hours.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Transfection, Flow Cytometry, Staining, Control

Journal: Journal of Oncology

Article Title: Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor

doi: 10.1155/2023/1453739

Figure Lengend Snippet: GCH1 inhibitor synergized with niraparib in reducing tumor burden without serious side-effects. (a) Representative images of tumors after niraparib and/or GCH1 inhibitor DAHP treatment in the PDX model. (b) Mean tumor volume of NSG mice subcutaneously implanted with PDX tumors. Tumor-bearing mice were dosed orally with vehicle or niraparib alone or in combination with DAHP. (c) Quantification of tumor weight from tumors described in (b). (d) Comparison of Ki67 expression between the four groups. (e) Quantification of mouse body weights. (f) Representative photos of HE staining of heart, liver, spleen, lung, kidney, and colon after niraparib and/or DAHP treatment. (g) Levels of ALT, AST, CK, CREA, and UREA in serum of the tumor-bearing mice.

Article Snippet: Cells were blocked with 5% BSA prior to staining with primary antibodies against GCH1 (A10616, Abclonal), γ -H2AX (GB111841, Servicebio, China), or RAD51 (GB11572, Servicebio, China) overnight, followed by incubation with appropriate secondary antibodies.

Techniques: Comparison, Expressing, Staining

Journal: ACS Omega

Article Title: Silencing of LncRNA XIST Suppressed Tumor Growth and Metastasis in Papillary Thyroid Carcinoma by Modulating miR-204/FN1 Axis

doi: 10.1021/acsomega.5c00390

Figure Lengend Snippet: Protein expression of EMT-related proteins (E-cadherin, N-cadherin, and Vimentin) in PTC cells with different transfections using Western blot. * p < 0.05, ** p < 0.01 compared with the blank group; # p < 0.05, ## p < 0.01 compared with the siNC + inhibitor NC group; and p < 0.05, and p < 0.01 compared with the si-lncRNA + inhibitor NC group; @ p < 0.05, @@ p < 0.01 compared with the si-lncRNA + miRNA inhibitor group; $ p < 0.05, $$ p < 0.01 compared with the siNC + miRNA inhibitor group; % p < 0.05, %%: p < 0.01 compared with the si-FN1 + inhibitor NC group.

Article Snippet: After blocking with 5% skim milk at 37 °C for 2 h, the membranes were incubated with anti-E-cadherin antibody (1:1000, Proteintech, Wuhan, China), anti-N-cadherin antibody (1:500, Boster, Wuhan, China), anti-Vimentin antibody (1:1000, Proteintech), and anti-GAPDH antibody (1:5000, Proteintech) overnight at 4 °C.

Techniques: Expressing, Transfection, Western Blot