Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 1. The miR-17-92 cluster members are dysregulated upon MMTV expression. (A) Heat-map of miR-17- 92 cluster upon small miRNAseq analysis of the normal mouse mammary epithelial cell lines, HC11 and HC11- MMTV. The miRNAseq was conducted on two biological replicates shown as 1 and 2. The numbers in the table represent the normalized reads observed upon miRNAseq. (B) Fold-change analysis of mature miR-17-92 cluster member expression using the miRNAseq data from HC11 cells. The Q-value (equivalent of p-value corrected for multiple test hypothesis) for each of the miRNA shown was statistically significant (Q < 0.005). (C) Western blot analysis of HEK293 and HEK293T cells expressing MMTV using an anti-MMTV Gag antibody. An antibody against a housekeeping gene (GAPDH) was used as a loading control. (D) Quantitative RT-PCRs (RT-qPCRs) to assess endogenous levels of the primary form of miR-17-92 cluster upon MMTV expression in HEK293T cells. b-Actin was used as the endogenous control. (E) RT-qPCR analysis of mature miR-17, miR-19a and miR-92a in HEK293T cells. U6 was used as the endogenous control. All experiments were carried out in triplicates. Statistical significance is shown as * where *p 0.05; **p 0.01, and ***p 0.001.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

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

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 2. Over-expression of the miR-17-92 cluster down-regulates MMTV expression. (A) An illustration of the miR-17-92 cluster, its truncated form miR-19a-20a-19b, and miR-92a over-expressed (OE) exogenously in the HEK293T-MMTV cells. (B) GFP-positive stably-transfected cells selected in puromycin and visualized using a fluorescent microscope (10x). (C) Expression of pri-miR-17-92 in miR-17-92 OE cell line quantified using a SYBR Green qPCR assay. (D) TaqMan miRNA assays used to quantify expression of mature miR-17, miR-19a, miR-92a in the miR17-92 OE cell line, (E) miR-19/20 OE cell line, and (F) miR-92a OE cell line. U6 snRNA was used as the endogenous control in these assays. (G) Western blot analysis of MMTV Gag expression in cell lysates. (H) MMTV RNA-specific TaqMan assay to quantify all MMTV messages in the three OE cell lines. (I) MMTV genomic RNA was assessed using gRNA-specific MMTV TaqMan assay in the miR-17-92 cluster OE and the miR-92a OE cells and virus particles isolated from culture supernatants (sup) via ultracentrifugation. The empty vector (EV) was designated as 1 in all analyses. Mean ± SD (n = 3). Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, ****p 0.0001. (J) Western blot analysis of viral particles harvested from culture supernatants in the respective miRNA OE stable cell lines.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Over Expression, Expressing, Stable Transfection, Transfection, Microscopy, SYBR Green Assay, Control, Western Blot, TaqMan Assay, Virus, Isolation, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 3. Mir-17-92 cluster and miR-92a inhibition rescues MMTV genomic RNA expression. TaqMan miRNA assays were used to quantify: (A) miR-17, miR-19a and miR-92a in miR-17-92 over expression (OE) cell line, (B) miR-19a in the miR-19a-20a-19b OE cell line, and (C) miR-92a in the miR-92a OE cell line. U6 snRNA was used as the endogenous control in the miRNA RT-qPCR assays. The error bars represent ± SD. NC, negative control scrambled oligo specific for each miRNA inhibitor used. (D) Western blot analysis of whole cell lysates (40 mg) from the miRNA OE cell lines treated with miRNA inhibitors for MMTV Gag expression. GAPDH was used as the internal control. Expression of the MMTV genomic RNA was quantified in the: (E) miR-17-92 OE cells treated with a cocktail of anti-miR-17, -19, & -92a oligos, (F) miR-19a- 20a-19b OE cells treated with anti-miR-19a oligos, and (G) miR-92a OE cells treated with anti-miR-92a oligos. b-Actin was used as the internal control in the MMTV TaqMan RT-qPCR assays. The empty vector (EV) was designated as 1 in all analyses. Mean ± SD (n = 3). (H) Western Blot analysis of whole cell lysates (50 mg) from the miR-92a OE cell line treated with anti-miR-92a oligos at varying concentrations. GAPDH was used as the internal control. Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, ****p 0.0001. ns, not significant; ns (P > 0.05).

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Inhibition, RNA Expression, Over Expression, Control, miRNA RT, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 4. Rescue of MMTV expression upon plasmid-based miRNA inhibition. (A) Mature miR-17 and (B) miR-92a levels were quantified in their respective PMIS inhibitor expressing cell lines using TaqMan miRNA RT- qPCRs. U6 snRNA was used as the endogenous control. (C) Expression of miR-17 target, PTEN was quantified using RT-qPCR; b-Actin was used as the internal control. (D) Western blot analysis of MMTV Gag expression across the three cell lines using 50 mg protein. GAPDH served as the endogenous control. (E) The expression of all MMTV transcripts were quantified using TaqMan RT-qPCRs with b-Actin as the internal control. The empty vector (EV; the PMIS backbone without any insert) was designated as unit 1 in all experiments with means represented as ±SD (n = 3). Statistical significance is shown as * * where *p 0.05 and **p 0.01.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Plasmid Preparation, Inhibition, miRNA RT, Control, Quantitative RT-PCR, Western Blot

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 5. MMTV expression rescued upon deletion or mutation of miR-92a seed sequence. (A) Schematic representation of the full miR-17-92 cluster (WT), or cluster with deletion of miR-92a (D92a) indicated as a dashed line, or a substitution mutation of miR-92a (92aMUT) within the cluster, both highlighted in red text. (B) Expression of MMTV Gag quantified by western blot analysis using 40 mg lysate from the different transiently transfected cell lines; GAPDH was used as the internal control. (C) Quantification of all MMTV mRNAs by RT-qPCR. b-Actin used as the internal control. The empty vector (EV) was designated as unit 1 with means represented as ± SD (n = 3). Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, and ****p 0.0001.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Mutagenesis, Sequencing, Western Blot, Transfection, Control, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 6. An in-silico analysis of the potential of the miR-17-92 cluster members to target the MMTV genomic RNA using STarMir bioinformatic tool. (A) Illustration of the MMTV genome and its various mRNAs along with the potential miR-17-92 cluster member predicted binding sites. The shaded box in blue highlights the region of the MMTV genome exclusively found in the full-length genomic RNA which is the same as the mRNA for Gag/Pro/Pol viral proteins. (B) Characterization of the miRNAs predicted to target the MMTV mRNAs. The target RNA regions highlighted in red belong exclusively to the full-length Gag/Pro/Pol mRNA or the genomic RNA (gRNA) with the number of target sites observed in parenthesis. (C) Illustration of the highest probable binding site of miR-92a on MMTV Gag.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: In Silico, Binding Assay

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 8. Expression of miR-17-92 cluster members in MMTV-induced tumors. Data representing the mean expression of miR-17-92 cluster members in MMTV-induced tumors (n = 2) and infected mammary glands (n = 2) obtained by small RNAseq analysis.58

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Expressing, Infection

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 9. Schematic representation of model representing miR-17-92-mediated regulation of MMTV gene expression. MMTV entry via murine transferrin receptor 1 (mTfR1) is followed by fusion of the viral capsid in a late endosomal compartment under low pH, uncoating/reverse transcription, and integration of the viral genome into the host chromosome. The cellular/viral factor-assisted transcriptional activation of the miR-17-92 cluster leads to up- regulation of the pri-miR-17-92. Once activated, due to the differential processing of the cluster, there is an increase in the expression levels of the mature miR-92a cluster member, which in turn, targets the MMTV unspliced genomic RNA. With more than 10 statistically significant miR-92a binding sites on the viral gag region, down-regulation of the genomic RNA takes place, resulting in suppression of MMTV replication. This is because the unspliced genomic RNA serves as the mRNA for the translation of the viral structural and enzymatic proteins (Gag/Pro/Pol) as well as the source of genomic RNA for encapsidation into the newly forming viral particles.Illustration created on BioRender.

Article Snippet: The blocked membranes were probed with either rabbit polyclonal MMTV anti-Gag antibody (Rockland Immunochemicals, Inc, Limerick, PA USA) at a dilution of 1:1000 in 2% non-fat dry milk overnight at 4 C, or anti-GAPDH followed by a 1-hour incubation at room temperature with the appropriate secondary antibody conjugated with horseradish peroxidase.

Techniques: Gene Expression, Reverse Transcription, Activation Assay, Expressing, Binding Assay

Journal: Virology Journal

Article Title: In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

doi: 10.1186/s12985-023-02242-5

Figure Lengend Snippet: Key resources and reagents

Article Snippet: Antibodies , Mouse Anti-HIV-CA-p24 (capture) , Sino Biological , 11695-MM08.

Techniques: Blocking Assay

Journal: Virology Journal

Article Title: In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

doi: 10.1186/s12985-023-02242-5

Figure Lengend Snippet: Key resources and reagents for the SB system

Article Snippet: Antibodies , Mouse Anti-HIV-CA-p24 (capture) , Sino Biological , 11695-MM08.

Techniques:

Journal: Virology Journal

Article Title: In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

doi: 10.1186/s12985-023-02242-5

Figure Lengend Snippet: Key resources and reagents for the ANG system

Article Snippet: Antibodies , Mouse Anti-HIV-CA-p24 (capture) , Sino Biological , 11695-MM08.

Techniques:

Journal: Virology Journal

Article Title: In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

doi: 10.1186/s12985-023-02242-5

Figure Lengend Snippet: Analyses of CA-p24 antigen concentration on diverse HIV isolates. Twenty-two HIV-1 isolates were tested through ABR-, ANG-, SB-, and RND-CA-p24 ELISAs for detection of CA-p24 antigen. All ELISAs detected most of the isolates, except RND-CA-p24 ELISA which did not detect the 92UG029 isolate. PBS was used as negative control. Values were measured in two independent ELISA runs and error bars represent the standard deviation (SD). The CA-p24 axis is log-scaled in the graph. The list of HIV isolates can be found in Table . ABR = Aalto Bio Reagents; ANG = Anogen; SB = Sino Biological; RND = R&D Systems

Article Snippet: Antibodies , Mouse Anti-HIV-CA-p24 (capture) , Sino Biological , 11695-MM08.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

Journal: Virology Journal

Article Title: In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

doi: 10.1186/s12985-023-02242-5

Figure Lengend Snippet: Fold-change in CA-p24 detection of HIV-1 A/B isolates relative to ABR-CA-p24 ELISA. ANG-CA-p24 ELISA exhibits low reactivity for HIV-1 A and B subtypes when compared to ABR-CA-p24 ELISA. RND-CA-p24 ELISA shows acceptable reactivity for HIV-1 A and B subtypes when compared to ABR-CA-p24 ELISA, but low reactivity for the SI22 isolate. SB-CA-p24 ELISA displays good reactivity for HIV-1 A and B subtypes when compared to ABR-CA-p24 ELISA, but low reactivity for the LAI isolate. ABR = Aalto Bio Reagents; ANG = Anogen; SB = Sino Biological; RND = R&D Systems

Article Snippet: Antibodies , Mouse Anti-HIV-CA-p24 (capture) , Sino Biological , 11695-MM08.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Virology Journal

Article Title: In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

doi: 10.1186/s12985-023-02242-5

Figure Lengend Snippet: Comparative reactivity towards HIV-1 isolates between assays as determined by Pearson’s r correlation. ANG-, SB-, and RND-CA-p24 ELISAs exhibit a significantly high correlation with ABR-CA-p24 ELISA. ANG-CA-p24 ELISA exhibits a significantly high correlation with RND-CA-p24 ELISA, whereas both RND- and ANG-CA-p24 ELISA show a low correlation with SB-CA-p24 ELISA. Concentrations of CA-p24 are displayed in ng/mL. ABR = Aalto Bio Reagents; ANG = Anogen; SB = Sino Biological; RND = R&D Systems

Article Snippet: Antibodies , Mouse Anti-HIV-CA-p24 (capture) , Sino Biological , 11695-MM08.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Virologica Sinica

Article Title: Altered hACE2 binding affinity and S1/S2 cleavage efficiency of SARS-CoV-2 spike protein mutants affect viral cell entry

doi: 10.1016/j.virs.2023.06.005

Figure Lengend Snippet: Expression and S1/S2 cleavage efficiency of SARS-CoV-2 variants' spike proteins. A Expression of SARS-CoV-2 variants' spike proteins in 293T cells. mCherry fluorescence intensity indicates spike protein expression, and GFP fluorescence intensity is the reference control. The fluorescence intensity ratio of mCherry/GFP indicates the relative expression level. The Y axis was normalized with S-WT fluorescence intensity ratio value. The numbers labeled on the bars indicate the average fold change of variants' spike protein expression relative to the S-WT. Omicron indicates BA.1 sublineage (also termed B.1.1.529) here. B , C Spike protein cleavage of pseudovirus (PVs) of the WT and variants probed with antibody against RBD. HIV-1 p24 was used to normalize the protein sample loading. D , E S1/S2 cleavage efficiency of SARS-CoV-2 spikes. The spike proteins of WT and variants were expressed in HEK293T cells and at 48 ​h post-transfection, cell lysates were made for Western blot analysis using the antibody against RBD. GAPDH served as the loading control. Full-length spike (S0) and S1 proteins are indicated. The ratio of S1 to the total spike was quantitatively analyzed using ImageJ. Data represent the mean ​± ​SD of 3–5 independent replicates. Significance was analyzed by one-way ANOVA. P values less than 0.05 were considered to be statistically significant. ∗∗∗∗, P ​< ​0.0001; ∗∗∗, P ​< ​0.001; ∗∗, P ​< ​0.01; ∗, P ​< ​0.05; ns, not significant ( P ​> ​0.05).

Article Snippet: The following primary antibodies were used: mouse anti-SARS-CoV-2 spike protein antibody (Sino Biological, Cat: 40592-MM117), mouse anti-HIV-1 p24 protein monoclonal antibody (Sino Biological, Cat: 11695-MM15), and mouse anti-GAPDH antibody (Abcam, ab8245).

Techniques: Expressing, Fluorescence, Control, Labeling, Transfection, Western Blot

Journal: Virologica Sinica

Article Title: Altered hACE2 binding affinity and S1/S2 cleavage efficiency of SARS-CoV-2 spike protein mutants affect viral cell entry

doi: 10.1016/j.virs.2023.06.005

Figure Lengend Snippet: Cell entry ability of SARS-CoV-2 pseudotyped variants bearing mutate spike proteins. A The ability of SARS-CoV-2 pseudotyped variants entering into 293T-hACE2 cells (only expressing hACE2), 293T-hACE2-TMPRSS2 cells (co-expressing hACE2 and TMPRSS2), Caco-2 ​cells (co-expressing hACE2 and TMPRSS2) and 293T cells (expressing low hACE2). B Entry comparison of pseudotyped variants in cells overexpressing hACE2 and TMPRSS2 or without TMPRSS2. HIV-1 p24 was used to normalize the PVs' additive does, and the data was calculated at 5 ​ng p24. The numbers labeled on the bars indicate ( A ) the average fold change of PV variants relative to the PV-WT or ( B ) the average fold change of 293T-hACE2-TMPRSS2 cells relative to 293T-hACE2 cells. Data are mean ​± ​SD of 3 independent replicates. Mock, uninfected control (no virus); P values less than 0.05 were considered to be statistically significant. ∗∗∗∗, P ​< ​0.0001; ∗∗∗, P ​< ​0.001; ∗∗, P ​< ​0.01; ∗, P ​< ​0.05; ns, not significant ( P ​> ​0.05). Significance was analyzed by ( A ) one-way ANOVA or ( B ) t -test.

Article Snippet: The following primary antibodies were used: mouse anti-SARS-CoV-2 spike protein antibody (Sino Biological, Cat: 40592-MM117), mouse anti-HIV-1 p24 protein monoclonal antibody (Sino Biological, Cat: 11695-MM15), and mouse anti-GAPDH antibody (Abcam, ab8245).

Techniques: Expressing, Comparison, Labeling, Control, Virus

Journal: Virologica Sinica

Article Title: Altered hACE2 binding affinity and S1/S2 cleavage efficiency of SARS-CoV-2 spike protein mutants affect viral cell entry

doi: 10.1016/j.virs.2023.06.005

Figure Lengend Snippet: Cross-species infection of SARS-CoV-2 pseudotyped variants bearing mutate spike proteins. A The ability of SARS-CoV-2 pseudotyped variants entering into cells expressing ACE2 orthologs of human, mouse, mouse, rat, rhesus, Odocoileus virginianus and with TMPRSS2 (blue columns) or without (orange columns). B A heatmap shows the fold change of the cross-species infection performance of PV variants. The Control group is the ability of PV-WT to enter into 293T-hACE2 cells. The color bar represents the scale. HIV-1 p24 was used to normalize the PVs' additive does, and the data was calculated at 5 ​ng p24. Data are mean ​± ​SD of 3–4 replicates (four for Mu, and three for others) and were normalized to the WT of the individual experiment. Mock, uninfected control (no virus); P values less than 0.05 were considered to be statistically significant. ∗∗∗∗, P ​< ​0.0001; ∗∗∗, P ​< ​0.001; ∗∗, P ​< ​0.01; ∗, P ​< ​0.05; ns, not significant ( P ​> ​0.05). Significance was analyzed by one-way ANOVA.

Article Snippet: The following primary antibodies were used: mouse anti-SARS-CoV-2 spike protein antibody (Sino Biological, Cat: 40592-MM117), mouse anti-HIV-1 p24 protein monoclonal antibody (Sino Biological, Cat: 11695-MM15), and mouse anti-GAPDH antibody (Abcam, ab8245).

Techniques: Infection, Expressing, Control, Virus