Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p predicted binding to mRNAs from ERK1 ( MAPK3 ) and ERK2 ( MAPK1 ). ( A ) Top 8 out of 1818 predicted targets of miR-483-5p from TargetScan 7.2 (based on context++ score) and the overlap with four experimentally-validated targets of miR-483-5p in miRTarBase 7.0. ( B ) Common targets predicted from TargetScan 7.2 for miR-483-5p and the neuroprotective miR-132-3p. ( C ) Table of predicted miRNA affinities for TAU ( MAPT ), ERK1 ( MAPK3 ) and ERK2 ( MAPK1 ) mRNAs in AD literature. LA = Luciferase assay, RT-qPCR = Reverse transcriptase quantitative PCR, IMB = Immunoblotting, N/A = Not available. The superscripted numbers indicate references where these interactions were verified.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p does not directly bind to TAU mRNA or impact TAU levels. ( A ) Scheme represents the predicted binding site for miR-483-5p in the 3′-UTR of the TAU mRNA. ( B ) Prediction indicates human-specific miR-483-5p binding site in 3′-untranslated region (3′-UTR) of the TAU mRNA among different species. ( C ) Quantitative reporter assay for luciferase activity in HEK293 cells. Reporter constructs carrying a single binding site (CTR, WT, MUT, PM) for miR-483-5p were tested. The miRNA activity on four pmirGLO constructs was measured simultaneously: an empty pmirGLO vector (CTR), a wild-type potential binding site for miR-483-5p (WT), a mutated binding site (MUT), and a site with full complementarity (PM). Interaction scheme of WT and MUT binding site of 3′-UTR of the TAU mRNA with miR-483-5p are shown below. PM (not shown here) is designed to perfectly match the miR-483-5p. Firefly luciferase activity was normalized against renilla luciferase activity. Data are shown as mean ± standard error from four independent experiments. One way ANOVA with post-hoc Tukey HSD was used for statistical analysis. ( D ) RT-qPCR analysis of TAU mRNA levels upon miR-483-5p mimic transfection in SK-N-MC cells. GAPDH was used as a reference. Data are shown as mean ± standard error from three biological replicates; Unpaired Student’s t -test was used for statistical analysis. In all cases, annotations were made on plots only where significant differences ( p < 0.05) were found.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Binding Assay, Reporter Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Quantitative RT-PCR, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p downregulates ERK1 at both the mRNA and protein level in HEK293 cells. ( A ) Scheme representing the predicted binding site for miR-483-5p in ERK1 and ERK2 3′-UTRs. ( B ) RT-qPCR analysis of ERK1 mRNA levels upon transfection with 10 nM and 50 nM of miR-483-5p mimic. GAPDH was used for normalization. ( C ) Immunoblot quantitative analysis of ERK1 protein levels upon transfection with miR-483-5p mimic at different concentrations. β-actin levels were used for normalization. ( D ) Representative immunoblot for ERK1/2 and pERK1/2 protein levels upon transfection with scramble and miR-483-5p mimic at 100 nM. ( E , F ) Immunoblot densitometry analysis of ERK1/2 and pERK1/2 protein levels upon transfection with scramble and miR-483-5p mimic at 100 nM. Data are shown as mean ± standard error from three independent experiments; Unpaired Student’s t -test was used for statistical analysis.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Binding Assay, Quantitative RT-PCR, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: CRISPR/Cas9-mediated knockout of miR-483-5p. ( A ) Scheme of the CRISPR/Cas9-mediated genomic deletion of miR-483-5p using upstream and downstream sgRNAs (sg1 and sg2). Fragment sizes of the wild-type allele (945 bp) and deleted region (212 bp). ( B ) Agarose gel electrophoresis of PCR amplified fragments (using primers shown in A ) from polyclonal cell populations retrieved upon transfection with empty plasmid without sgRNA (Cas9 only) and Cas9 +/sgRNA expressing plasmid (Cas9 + miR-483-5p sgRNA). 733 bp band corresponds to deleted allele. ( C ) Fold change decrease in miR-483-5p levels in CRISPR/Cas9-deleted cells (Cas9 + miR-483-5p sgRNAmiR-483 KO). miR-191-5p was used for normalization. ( D ) Fold change increase in ERK1 mRNA ( MAPK3 ) levels in CRISPR/Cas9-deleted cells. GAPDH was used for normalization. Data represent the mean ± standard deviation from two independent experiments. Statistical analysis was performed using an unpaired Student’s t -test on log 2 (fold change).

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: CRISPR, Knock-Out, Agarose Gel Electrophoresis, Amplification, Transfection, Plasmid Preparation, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p mediated downregulation of total and phosphorylated ERK1/2 correlates with reduced levels of phosphorylated TAU in HEK293 cells expressing EGFP-TAU and upon transfection with scramble and miR-483-5p mimics at 100 nM. ( A ) Representative immunoblots of total and phosphorylated levels of TAU, ERK1 and ERK2 under the different experimental conditions. β-actin was used as loading control. ( B ) Quantitative analysis of the immunoblot levels for TAU, ERK1 and ERK2. ( C ) Quantitative analysis of the immunoblot levels for pTAU, pERK1 and pERK2. Data are shown as mean ± standard error of three independent experiments. One way ANOVA with post-hoc Tukey HSD was used and statistically significant differences are shown with p values.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Expressing, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p-mediated downregulation of phosphorylated ERK1/2 is associated with a trend in decreased levels of phosphorylated TAU in SK-N-MC cells. ( A ) Representative immunoblots of total and phosphorylated levels of TAU, ERK1 and ERK2 in untransfected (mock) or transfected SK-N-MC cells with 100 nM miR-483-5p mimic. C q values for miR-483-5p transfected cells are shown and in mock miR-483-5p was undetermined (Und). β-actin was used as loading control. ( B ) Quantitative analysis of the immunoblot levels for TAU, ERK1 and ERK2. ( C ) Quantitative analysis of the immunoblot levels for pTAU, pERK1 and pERK2. Data are shown as mean ± standard error of the mean from four biological replicates; Unpaired Student’s t -test was used to compare groups and we observed no statistically significant differences.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Western Blot, Transfection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

doi: 10.3389/fcimb.2023.1098420

Figure Lengend Snippet: Chlamydia infection induces expression of a subset of YAP target genes. (A) Volcano plot of gene expression in bulk RNA-sequencing of End1/E6E7 immortalized epithelial cells (End1s) during infection with Chlamydia trachomatis ( Ct ) serovar L2 compared to mock infection, at 24 hours post-infection (hpi). n = 3, with a minimum of 3x10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes whose expression differed most significantly (lowest FDRP) from the mock infection. (B) Table of selected transcription factors identified as potential targets of infection-associated modulation via cross-referencing of differentially expressed genes identified in (A) with the ChIP Enrichment Analysis (ChEA) database of transcription factor target genes. See also Supplementary Data S2. (C) Volcano plot of gene expression in bulk RNA-sequencing of primary human endocervical epithelial cells (HCECs) during infection with Ct serovar L2 compared to mock infection, at 24 hours post-infection (hpi). n = 3, with a minimum of 3x10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes whose expression differed most significantly (lowest FDRP) from the mock infection. (D) Venn diagram of differentially expressed genes identified in (A, C) cross-referenced with the ChEA database of YAP target genes. (E) Scatter plot of gene expression of YAP-responsive (ChEA), differentially expressed genes in either Ct serovar L2-infected End1s (x-axis) or HCECs (y-axis). All fold changes are relative to each cell type’s respective mock-infected control; blue line: linear regression model of correlation; grey shading: 95% confidence interval. R 2 and p-values calculated using Pearson’s correlation. (F) Heatmap of YAP target gene expression in Ct serovar L2-infected End1s (left columns) and HCECs (right columns). All fold changes are relative to each cell type’s respective mock-infected control; only genes differentially expressed (FDRP ≤ 0.05) in both cell types are shown. (G) Dot plot of GO biological process term enrichment in the set of YAP target genes differentially expressed in Ct serovar L2 infection of End1s or HCECs (top 25 most significantly enriched terms shown). Dot size: number of term-associated genes found in set, dot color: adjusted p-value.

Article Snippet: Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO 2 in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042).

Techniques: Infection, Expressing, Gene Expression, RNA Sequencing, Control, Targeted Gene Expression

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

doi: 10.3389/fcimb.2023.1098420

Figure Lengend Snippet: Chlamydia infection promotes YAP nuclear translocation. (A) Expression of CTGF and CYR61 at 24 hpi in mock- and Ct L2-infected End1 cells, as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; n.s.: not significant (p-values > 0.05), asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (B) Expression of CTGF at 24 hpi in mock- and Ct L2-infected End1 cells transfected with non-targeting (NT) or YAP1-targeting siRNA (10 nM for 24 h prior to infection), as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (C) Expression of CTGF, INHBA, and BMP2 at 24 hpi in mock- and Ct L2-infected End1 cells treated with the YAP-TEAD inhibitor verteporfin (Vpf, 5 μM for 16 h starting at 8 hpi), as measured by RT-qPCR. n = 5 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (D) Representative micrographs of YAP (green) translocation into the nuclei (blue) of confluent mock- and Ct L2-infected End1 cells at 24 hpi. Asterisks: chlamydial inclusions, scale bar: 20 μm. (E) Quantification of YAP nuclear translocation in (D) as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (F) Representative micrographs of YAP nuclear translocation of confluent mock- and Ct L2-infected primary human cervical epithelial cells at 24 hpi. Asterisks, chlamydial inclusions, scale bar: 20 μm. (G) Quantification of YAP nuclear translocation in (F) as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (H) Quantification of YAP nuclear translocation at 2, 4, 8, 12, 18, and 24 hpi in confluent mock- and Ct L2-infected End1 cells as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Blue dots: mock-infected cells, red dots: Ct L2-infected cells, black bars: group means, asterisks: p-value ≤ 0.05, using pairwise Wilcoxon rank-sum tests and Bonferroni’s correction for multiple comparisons. (I) Representative micrographs of YAP nuclear translocation at 18 hpi in confluent mock- and Ct L2-infected End1 cells treated with chloramphenicol (Cm, 50 μg/mL for 1 h at 17 hpi) or DMSO. Asterisks: chlamydial inclusions; scale bar: 20 μm. (J) Quantification of YAP nuclear translocation in (I) . n = 3 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank-sum tests and Bonferroni’s correction for multiple comparisons.

Article Snippet: Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO 2 in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042).

Techniques: Infection, Translocation Assay, Expressing, Quantitative RT-PCR, Control, Transfection, Fluorescence

Journal: Bioengineered

Article Title: Circular RNA circ_0000228 promotes the malignancy of cervical cancer via microRNA-195-5p/ lysyl oxidase-like protein 2 axis.

doi: 10.1080/21655979.2021.1954846

Figure Lengend Snippet: Figure 1. Circ_0000228 was remarkably up-modulated in CC tissues and cell lines. (a) A volcano plot was employed to analyze the differentially expressed circRNAs in the GEO dataset (GSE113696) that were remarkably up- and down-modulated in CC cell lines, compared with normal cervical epithelial cells; (b) A heat maps was utilized to present the significantly highly expressed circRNAs in CC; (c) Circ_0000228 expression in 40 CC tissues/paired adjacent cervical tissues was examined by qRT-PCR; (d) Circ_0000228 expression in 21 patients with stage I CC and 19 patients with stage II CC was analyzed by qRT-PCR, respectively; (e) ZEB1 mRNA expression in 21 patients with stage I CC and 19 patients with stage II CC was analyzed by qRT-PCR, respectively; (f) Circ_0000228 expression in HUCEC and CC cell lines (HeLa, HCC94, SW756, C33A) was detected by qRT-PCR; (g) Circ_0000228 expression in the nucleus and cytoplasm of HCC94 and C33A cells was detected by qRT-PCR. All of the experiments were performed in triplicate. ***P < 0.001.

Article Snippet: Four human CC cell lines (HeLa, HCC94, SW756, and C33A) and human normal cervical epithelial cells (HUCEC) were procured from the American Type Culture Collection (ATCC, Bethesda, MD, USA) and the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Circular RNA circ_0000228 promotes the malignancy of cervical cancer via microRNA-195-5p/ lysyl oxidase-like protein 2 axis.

doi: 10.1080/21655979.2021.1954846

Figure Lengend Snippet: Figure 3. Circ_0000228 modulated LOXL2 expression through miR-195-5p. (a) A volcano plot was adopted to show the miRNAs that were remarkably up- and down-modulated in CC tissues compared with normal cervical tissue in the GEO dataset (GSE86100); (b) The Venn diagram was used to screen out the differentially expressed miRNAs in GSE86100, which contained complementary binding sites with circ_0000228; (c) Bioinformatics analysis indicated that high LOXL2 expression suggested poor clinical outcome of CC patients. (d) The complementary binding sites of circ_0000228 and miR-195-5p, miR-195-5p and LOXL2 3UTR are presented, and the corresponding luciferase reporter vectors were designed; (E-F) Dual-luciferase reporter gene experiments confirmed that the binding sites between circ_0000228 and miR-195-5p, miR-195-5p and LOXL2 3UTR were functional. (g) MiR-195-5p expression in 40 pairs of CC tissues/adjacent cervical tissues was detected by qRT-PCR; (h) MiR-195-5p expression in stage I and stage II CC patients was detected by qRT-PCR, respectively; (I) MiR-195-5p in HUCEC and CC cell lines (HeLa, HCC94, SW756, C33A) was detected by qRT-PCR.(j) LOXL2 mRNA expression in 40 pairs of CC tissues/adjacent cervical tissues was detected by qRT-PCR; (k) LOXL2 mRNA expression in HUCEC and CC cell lines (HeLa, HCC94, SW756, C33A) was detected by qRT-PCR; (L) LOXL2 mRNA expression in stage 1 and stage 2 CC patients was detected by qRT-PCR; (m-o) Pearson’s correlation analysis of the correlation relationships among circ_0000228 miR-195-5p, and LOXL2 expression in CC tissues; (p) qRT- PCR was employed to detect miR-195-5p expression after co-transfection with circ_0000228 siRNA and miR-195-5pin in CC cell lines; (q) qRT-PCR and Western blot experiments were employed to detect LOXL2 expression after co-transfection with circ_0000228 siRNA and miR-195-5p inhibitors in CC cell lines; All of the experiments were performed in triplicate. *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: Four human CC cell lines (HeLa, HCC94, SW756, and C33A) and human normal cervical epithelial cells (HUCEC) were procured from the American Type Culture Collection (ATCC, Bethesda, MD, USA) and the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank (Shanghai, China).

Techniques: Expressing, Binding Assay, Luciferase, Functional Assay, Quantitative RT-PCR, Cotransfection, Western Blot

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: Quantitative Temporal Analysis of VACV Infection (A) Schematic of experimental workflow for each of three biological replicates. Cells were infected at MOI of 5 or mock infected ( Figure S1 A). Additionally, one mock and one infected sample were treated for 6 h with the viral DNA replication inhibitor cytosine arabinoside (AraC). (B) Hierarchical cluster analysis of all proteins quantified. An enlargement of three subclusters is shown (right panel), including multiple proteins that were substantially up- or downregulated. (C) Scatterplot of all proteins quantified at 18 h of infection. For all analyses in this manuscript, a mean fold change at each time point was calculated by averaging fold changes from each of the biological replicates in which the protein was quantified. For the purposes of comparison, the 18-h mock sample from each replicate was used, because the 0-, 6-, and 18-h mock samples behaved extremely similarly ( Figure S1 E). To perform a comprehensive analysis, “sensitive” criteria were employed, examining proteins down- or upregulated >2-fold on average across all replicates in which the protein was quantified ( A). Sensitive criteria were used in each analysis apart from where indicated. Data from “stringent” criteria that examined only proteins quantified in all three replicates with an average fold change > 2 and p < 0.05 are shown in C. For proteins quantified in all three replicates, a Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. Only proteins quantified in all three replicates are shown in this scatterplot. (D) Example of a previously unreported target of VACV infection. Data are represented as mean ± SEM; ∗ p < 0.05 (see ). Immunoblot of HFFF-TERTs infected with VACV (MOI = 5) confirmed rapid HDAC5 downregulation.

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Infection, Comparison, Two Tailed Test, Western Blot

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: VACV Protein C6 Downregulates HDAC5 (A) HDAC5, but not HDAC1, is proteasomally degraded during VACV infection. Bar charts and statistics were generated as described in Figure 5 . (B) Viral class centroid profiles compared to an inverted profile of HDAC5, which had additionally been scaled from 0 to 1. (C) Profile of HDAC5 scaled as in (B), and Tp2-class VACV proteins with known roles in regulation of IFN or ISGs. (D) C6 targets HDAC5. HFFF-TERTs were infected in biological triplicate with WT VACV or vΔC6 (lacking gene C6L ) ( Unterholzner et al., 2011 ) (MOI = 5 and 12 h). The scatterplot shows all proteins quantified. A Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. (E) Representative immunoblot demonstrating that C6 specifically targets HDAC5. By comparison, HDAC1 was unmodified (MOI = 5). Quantitation of all three replicate immunoblots are shown (right panel). Data are represented as mean ± SEM, ∗ p < 0.05 from a two-tailed t test. (F) Representative immunoblot demonstrating rescue of HDAC5 expression by inhibition of the proteasome (MOI = 5). MG132 was added 2 h after VACV infection. Quantitation of all three replicate immunoblots is shown (right panel). Data are represented as mean ± SEM, ∗∗ p < 0.00005 from a two-tailed t test. (G) Immunoblot demonstrating that inducible expression of C6 is sufficient for HDAC5 degradation. HEK293T cells inducibly expressing C6 with an N-terminal Myc or C-terminal TAP tag were either treated or untreated with 100 ng/ml doxycycline overnight then lysates immunoblotted.

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Infection, Generated, Two Tailed Test, Western Blot, Comparison, Quantitation Assay, Expressing, Inhibition

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet:

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Virus, Western Blot, Derivative Assay, Subcloning, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, TA Cloning, Sequencing, Mass Spectrometry, CRISPR, Disruption, Plasmid Preparation, Software, Quantitative Proteomics, Imaging, Flow Cytometry, Fluorescence, Microscopy

Journal: American Journal of Translational Research

Article Title: Extracellular vesicles from human umbilical cord mesenchymal stem cells treated with siRNA against ELFN1-AS1 suppress colon adenocarcinoma proliferation and migration

doi:

Figure Lengend Snippet: ELFN1-AS1 expression is up-regulated in colon adenocarcinoma (COAD). A. Hierarchical cluster heat map of differentially expressed lncRNAs in COAD and corresponding normal tissues generated from RNA sequencing data from the TCGA database. Red in the heat map denotes upregulation; green denotes downregulation. The red arrow indicates ELFN1-AS1. B. Expression of ELFN1-AS1 in the GEPIA database. *P<0.05 vs. Normal group (Mann-Whitney test). C. Kaplan-Meier survival analysis of COAD patients’ overall survival based on ELFN1-AS1 expression in GEPIA. D. The mRNA level of ELFN1-AS1 was determined by qRT-PCR in 22 pairs of tumor tissues and adjacent non-tumor tissues. *P<0.05 vs. non-tumor group (Mann-Whitney test). E. ELFN1-AS1 expression in COAD cell lines (CaCO-2, SW-480, HCT-116) compared with normal colorectal epithelial cells FHC detected by qRT-PCR. **P<0.01 vs. FHC group (one-way analysis of variance).

Article Snippet: Cell culture The human COAD cell lines (CaCO-2, SW-480 and HCT-116) and normal colorectal epithelial cells FHC were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Generated, RNA Sequencing, MANN-WHITNEY, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: Extracellular vesicles from human umbilical cord mesenchymal stem cells treated with siRNA against ELFN1-AS1 suppress colon adenocarcinoma proliferation and migration

doi:

Figure Lengend Snippet: Down-regulation of ELFN1-AS1 expression inhibits COAD cell proliferation. A, B. The expression levels of ELFN1-AS1 mRNA in CaCO-2 and HCT-116 siRNA group were detected by real-time PCR. **P<0.05 and ***P<0.001 vs. NC group (one-way analysis of variance). C, D. CaCO-2 and HCT-116 cell lines transfected with si-RNA or si-NC were cultured in 96-well plates, cell proliferation were measured via CCK-8. *P<0.05, **P<0.05 and ***P<0.001 vs. NC group (one-way analysis of variance). E. The colony forming growth assay was performed to determine the proliferation of siRNA/si-NC transfected COAD cells.

Article Snippet: Cell culture The human COAD cell lines (CaCO-2, SW-480 and HCT-116) and normal colorectal epithelial cells FHC were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Cell Culture, CCK-8 Assay, Growth Assay

Journal: American Journal of Translational Research

Article Title: Extracellular vesicles from human umbilical cord mesenchymal stem cells treated with siRNA against ELFN1-AS1 suppress colon adenocarcinoma proliferation and migration

doi:

Figure Lengend Snippet: Down-regulation of ELFN1-AS1 gene expression inhibits COAD cell migration. A. A wound healing assay was applied to analyze the migration capacity in COAD cells after transfection with siRNA or NC. B, C. The quantifications of COAD cell migration were presented as percentage the ratios of closure. **P<0.05 vs. NC group (one-way analysis of variance). D. Transwell migration assay in down-regulation of ELFN1-AS1 cells and their corresponding control cells. E. Image J software was used for cell counting and the columns represent the mean of cell numbers from at least three independent experiments. ***P<0.001 vs. NC group (one-way analysis of variance).

Article Snippet: Cell culture The human COAD cell lines (CaCO-2, SW-480 and HCT-116) and normal colorectal epithelial cells FHC were purchased from ATCC (Manassas, VA).

Techniques: Gene Expression, Migration, Wound Healing Assay, Transfection, Transwell Migration Assay, Control, Software, Cell Counting

Journal: American Journal of Translational Research

Article Title: Extracellular vesicles from human umbilical cord mesenchymal stem cells treated with siRNA against ELFN1-AS1 suppress colon adenocarcinoma proliferation and migration

doi:

Figure Lengend Snippet: Down-regulation of ELFN1-AS1 regulates the EMT in COAD cell lines. A. The effect of ELFN1-AS1 expression on the levels of Erk and p-Erk. B. Protein expression of Erk and p-Erk. ***P<0.001 vs. NC group (one-way analysis of variance). C. The effect of ELFN1-AS1 expression on the levels of E-cadherin, and Vimentin in COAD cell lines transfected with si-RNA or si-NC by Western blot. D. Protein expression of E-cadherin, and Vimentin were analyzed by Image J software. *P<0.05, **P<0.05 and ***P<0.001 vs. NC group (one-way analysis of variance).

Article Snippet: Cell culture The human COAD cell lines (CaCO-2, SW-480 and HCT-116) and normal colorectal epithelial cells FHC were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Western Blot, Software

Journal: American Journal of Translational Research

Article Title: Extracellular vesicles from human umbilical cord mesenchymal stem cells treated with siRNA against ELFN1-AS1 suppress colon adenocarcinoma proliferation and migration

doi:

Figure Lengend Snippet: SiRNA-EVs inhibit COAD cell proliferation and migration in vitro. A, B. The expression of ELFN1-AS1 in COAD cells treated with siRNA-EVs (200 μg/ml for 72 h) was detected by real-time PCR. *P<0.05 and **P<0.01 vs. NC-EV group (one-way analysis of variance). C. Erk and p-Erk protein were detected by western blotting after treatment by siRNA-EVs in COAD cells. D. EMT relevant proteins (E-cadherin and Vimentin) were detected by western blotting analysis after treatment by siRNA-EVs in COAD cells. E. Colon forming assays were used to determine the colony-forming ability of COAD cells after siRNA-EVs treatment. F. Transwell migration assay in siRNA-EVs treated-COAD cells and their corresponding control cells.

Article Snippet: Cell culture The human COAD cell lines (CaCO-2, SW-480 and HCT-116) and normal colorectal epithelial cells FHC were purchased from ATCC (Manassas, VA).

Techniques: Migration, In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transwell Migration Assay, Control

Journal: Cancer Genomics & Proteomics

Article Title: RFC3 Knockdown Decreases Cervical Cancer Cell Proliferation, Migration and Invasion

doi: 10.21873/cgp.20493

Figure Lengend Snippet: Differential expression of RFC3 in cervical cancer tissues and cell lines. (A) RFC3 mRNA expression in cervical cancer tissues (n=306) and normal tissues (n=13), in addition to overall survival and disease-free survival were analyzed using the GEPIA2 web tool. (B) RFC3 mRNA expression in cervical cancer cell lines and human primary cervical epithelial cell was assessed using RT-qPCR analysis. The experiment was performed three times, and significant differences were determined by mean±SD. **p<0.01, ***p<0.001. (C) RFC3 protein expression was detected by western blotting in the same cell lines. Cell lysates were immunoblotted with an anti-RFC3 antibody, and an anti-β-actin antibody was used as a loading control.

Article Snippet: Human primary cervical epithelial cells were cultured in cervical epithelial cell basal medium added the contents of cervical epithelial cell growth kit (ATCC).

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Control