Journal: Molecular Therapy

Article Title: Intra-articular Delivery of Antago-miR-483-5p Inhibits Osteoarthritis by Modulating Matrilin 3 and Tissue Inhibitor of Metalloproteinase 2

doi: 10.1016/j.ymthe.2016.12.020

Figure Lengend Snippet: miR-483-5p Directly Targets Matn3 in Chondrocytes during OA Development

Article Snippet: Intra-articular Injection With regard to lentivirus (GenePharma) injection, 10 μL lentivirus-mediated miR-483-5p (5′-AAGACGGGAGAAGAGAAGGGAG-3′) (4 × 10 8 TU/ml), siTimp2 (5′- GGAATGACATCTATGGCAA-3′) (1 × 10 9 TU/mL), or NC (5′- TTCTCCGAACGTGTCACGTTTC-3′), Matn3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010770.4","term_id":"306518577","term_text":"NM_010770.4"}} NM_010770.4 ) (Cyagen Biosciences), TIMP2( {"type":"entrez-nucleotide","attrs":{"text":"NM_011594.3","term_id":"61835847","term_text":"NM_011594.3"}} NM_011594.3 ) (Cyagen Biosciences), and corresponding negative controls were injected into the knee joint 34 of male mice (n = 5/group) using a 33G needle and a micro-syringe (Hamilton).

Techniques:

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: HPV E2 interacts with Pyk2. (A) HEK293TT cells were transfected with GFP-Pyk2 WT (wild type) or GFP-Pyk2 KD (kinase dead) and FLAG-HPV-31 E2. FLAG-HPV-31 E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (B) HEK293TT cells were transfected with GFP-Pyk2 WT and FLAG-HPV-16 E2. FLAG-HPV-16 E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (C) HPV-16 E2 in W12 cells was immunoprecipitated using sheep anti-HPV-16 E2 serum or normal sheep IgG. Complexes were blotted with Pyk2 and HPV-16 E2 (TVG-261) antibodies.

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Pyk2 interacts with the TAD of E2. (A) Illustration of HPV-31/-16 E2 fragments. (B) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2 or HPV-31 E2 fragments (aa 107 to 372, 176 to 372, and 205 to 372) and immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (C) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2 or HPV-31 E2 with the β-sheet region of the E2 TAD deleted (aa 1 to 106 and aa 176 to 372) and immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (D) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-16 E2 or HPV-16 E2 fragments (aa 1 to 104, 202 to 365, 105 to 365, and 202 to 365). After immunoprecipitation with M2 (FLAG) antibodies, complexes were blotted with M2 (FLAG) and Pyk2 antibodies.

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Pyk2 phosphorylates the E2. (A) HEK293TT cells were transfected with GFP-Pyk2 WT or GFP-Pyk2 KD and FLAG-HPV-31 or FLAG-HPV-16 E2. FLAG-HPV-E2 was immunoprecipitated with M2 (FLAG) antibodies, and complexes were blotted with pTyr-1000, M2 (FLAG), and Pyk2 antibodies. (B) HEK293TT cells were transfected with FLAG-Pyk2 WT or FLAG-Pyk2 KD and FLAG-HPV-31 E2 fragments (aa 1 to 106, 176 to 372, 107 to 372, and 205 to 372). FLAG-HPV-31 E2 and FLAG-Pyk2 were immunoprecipitated with M2 (FLAG) antibodies, and complexes were blotted with pTyr-1000 and M2 (FLAG) antibodies. Phospho-E2 fragments are identified by an asterisk (*).

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Pyk2 induced tyrosine phosphorylation of HPV-31 E2 Y to F mutants. HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2 phenylalanine F mutants (A) or with GFP-Pyk2 WT or GFP-Pyk2 KD and FLAG-HPV-31 F mutants (B). Lysates were immunoprecipitated with M2 (FLAG), and bound proteins were blotted with Pyk2, pTyr-1000, and M2 (FLAG) antibodies. (C) Quantification of phosphotyrosine levels of Y131F. Values are means ± the SEM (n = 3). *, P < 0.05 (two-way t test).

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Nuclear localization of HPV E2 and Pyk2. (A) CV-1 cells were transfected with GFP-Pyk2 WT and FLAG-HPV-31 or -16 E2 constructs. Immunofluorescence staining for HPV-E2 proteins with M2 (FLAG) antibodies and DAPI (blue) was performed. Cells were visualized under ×60 magnification using confocal microscopy. Pyk2 (Green), E2 (red), and DAPI (blue) are indicated. (B) HeLa cells were transfected with FLAG-HPV-31 E2 WT or Y131 mutants. Immunofluorescence staining was done with M2 (FLAG, red) antibodies or DAPI (blue). Magnification, ×60.

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Transfection, Construct, Immunofluorescence, Staining, Confocal Microscopy

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Pyk2 siRNA increases HPV DNA content. (A) CIN612-9E cells were transfected with a scrambled control, Pyk2, or EGFR siRNA. After 72 h, lysates were immunoblotted with Pyk2, EGFR, or β-actin antibodies. CIN612-9E cells were transfected with a control, Pyk2, or EGFR siRNA. Real-time PCR was performed for the HPV-31 long control region (LCR) and normalized to the level of β-actin. Values are expressed as means ± the SEM (n = 6). *, P < 0.05. (B) W12 cells were transfected with a control, Pyk2, or EGFR siRNA. After 72 h, the lysates were immunoblotted with Pyk2, EGFR, and β-actin antibodies. W12 cells were transfected with a control, Pyk2, or EGFR siRNA. DNA was isolated for real-time PCR for the HPV16 E6 DNA region and normalized to the levels of β-actin. Values are expressed as means ± the SEM (n = 12). *, P < 0.05.

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Isolation

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Pyk2 inhibition increases HPV DNA content by decreasing E2 tyrosine phosphorylation. (A) CIN612-9E and W12 cells were treated with the Pyk2 inhibitor PF-431396 for 24 h, and cell viability was quantified by an MTS cell proliferation assay. Values are expressed as means ± the SEM (n = 8). *, P < 0.05. (B) CIN612-9E cells were incubated with 100 nM PF-431396, and W12 cells were incubated with 10 or 100 nM PF-431396 for 72 h. DNA was isolated, and real-time-PCR was performed for HPV-31 or HPV-16 DNA region near the LCR and normalized to the levels of β-actin. Values are expressed as means ± the SEM (n = 6). *, P < 0.05. (C) CIN612-9E cells were treated with 10 or 100 nM PF-431396 for 72 h. RNA was isolated and converted to cDNA using reverse transcription-PCR. Real-time PCR was carried out with primers to HPV-31 E1̂E4 and HPV-31 E2̂E8 mRNA and normalized to 18S transcripts. Values are means ± the SEM (n = 3). *, P < 0.05. (D) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2. Cells were treated with 10 or 100 nM PF-431396 for 24 h. E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with pTyr-1000, Pyk2, and M2 (FLAG) antibodies.

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Inhibition, Phospho-proteomics, Proliferation Assay, Incubation, Isolation, Real-time Polymerase Chain Reaction, Reverse Transcription, Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

doi: 10.1128/JVI.01110-20

Figure Lengend Snippet: Pyk2 knockout increases HPV genome integration. (A) CIN612-9E cells infected with pLentiCRISPR v2 (control) or pLentiCRISPR v2-Pyk2 guide constructs. Lysates were immunoblotted with Pyk2 and β-actin antibodies. (B) DNA from control and Pyk2 knockout (KO) CIN612-9E cell lines were analyzed by qPCR for the HPV-31 LCR and normalized to β-actin. Values are expressed as means ± the SEM (n = 9). (C) DNA from control and Pyk2 KO CIN612-9E cell lines were subjected to exonuclease V digestion. Resistant HPV-31 DNA was quantified by qPCR. Actin DNA was used as a positive control for digestion, and mitochondrial DNA was used as a resistant control. Values are expressed as means ± the SEM (n = 9) *, P < 0.05. (D). Real-time PCR was performed using primers for HPV-31 E1̂E4, E8̂E2, and E6 transcripts and normalized to actin transcripts. Values are means ± the SEM (n = 3). *, P < 0.05.

Article Snippet: Y131 was mutated in pCDNA3-HPV-31 E2 using the Q5 site-directed mutagenesis kit (New England BioLabs) and confirmed by sequencing. pLentiCRISPR v2 was from Addgene (catalog no. 52961) ( 43 ), and pLentiCRISPR v2 human Pyk2 guide 1 was from GenScript.

Techniques: Knock-Out, Infection, Control, Construct, Positive Control, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy

Article Title: Intra-articular Delivery of Antago-miR-483-5p Inhibits Osteoarthritis by Modulating Matrilin 3 and Tissue Inhibitor of Metalloproteinase 2

doi: 10.1016/j.ymthe.2016.12.020

Figure Lengend Snippet: miR-483-5p Directly Targets Timp2 in Chondrocytes In Vitro and in Mice

Article Snippet: Intra-articular Injection With regard to lentivirus (GenePharma) injection, 10 μL lentivirus-mediated miR-483-5p (5′-AAGACGGGAGAAGAGAAGGGAG-3′) (4 × 10 8 TU/ml), siTimp2 (5′- GGAATGACATCTATGGCAA-3′) (1 × 10 9 TU/mL), or NC (5′- TTCTCCGAACGTGTCACGTTTC-3′), Matn3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010770.4","term_id":"306518577","term_text":"NM_010770.4"}} NM_010770.4 ) (Cyagen Biosciences), TIMP2( {"type":"entrez-nucleotide","attrs":{"text":"NM_011594.3","term_id":"61835847","term_text":"NM_011594.3"}} NM_011594.3 ) (Cyagen Biosciences), and corresponding negative controls were injected into the knee joint 34 of male mice (n = 5/group) using a 33G needle and a micro-syringe (Hamilton).

Techniques: In Vitro

Journal: Molecular Therapy

Article Title: Intra-articular Delivery of Antago-miR-483-5p Inhibits Osteoarthritis by Modulating Matrilin 3 and Tissue Inhibitor of Metalloproteinase 2

doi: 10.1016/j.ymthe.2016.12.020

Figure Lengend Snippet: miR-483-5p Downregulates Timp2 to Promote Cartilage Angiogenesis and OA Development

Article Snippet: Intra-articular Injection With regard to lentivirus (GenePharma) injection, 10 μL lentivirus-mediated miR-483-5p (5′-AAGACGGGAGAAGAGAAGGGAG-3′) (4 × 10 8 TU/ml), siTimp2 (5′- GGAATGACATCTATGGCAA-3′) (1 × 10 9 TU/mL), or NC (5′- TTCTCCGAACGTGTCACGTTTC-3′), Matn3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010770.4","term_id":"306518577","term_text":"NM_010770.4"}} NM_010770.4 ) (Cyagen Biosciences), TIMP2( {"type":"entrez-nucleotide","attrs":{"text":"NM_011594.3","term_id":"61835847","term_text":"NM_011594.3"}} NM_011594.3 ) (Cyagen Biosciences), and corresponding negative controls were injected into the knee joint 34 of male mice (n = 5/group) using a 33G needle and a micro-syringe (Hamilton).

Techniques: