primary antibodies against collagen type ii alpha 1  (Servicebio Inc)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: Primer sequences for qRT-PCR.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Sequencing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: USP7 knockdown inhibits ATDC5 cell proliferation and increases apoptosis and inflammatory response after 48 h chondrogenic induction under TNF- α -induced inflammation. (a) Relative USP7 mRNA expression under 20 ng/mL TNF- α . (b) Relative USP7 protein expression in USP7 knockdown and its control groups. (c) Alcian blue and toluidine blue staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. Scale bars = 100 μ m. (d) Growth curves in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction measured by CCK-8 assay. (e) Relative Col2a1 and Sox9 mRNA expression of in USP7 knockdown and its control groups under TNF- α stimulation after 48 h chondrogenic induction. (f) Col2a1, Sox9, Cleaved Caspase-3, Bax, and Bcl-2 protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. (g) Quantitative measurement of (f). (h) Relative Caspase-3 activity in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. (i) Quantitative measurement of cell apoptosis measured by flow cytometry in the USP7-nc and USP7-sh2 groups under TNF- α -induced inflammation after 48 h chondrogenic induction. (j) TUNEL staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. White arrows indicated TUNEL-positive cells. Scale bars = 50 μ m. (k) Quantitative measurement of (j). (l) Relative IL-6 , COX , NOS2 , and MMP13 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. (m) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining, CCK-8 Assay, Activity Assay, Flow Cytometry, TUNEL Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: ERS signaling inhibitor 4-PBA reverses chondrocyte proliferation, apoptosis, and inflammation caused by USP7 knockdown under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (c) Quantitative measurement of (b). (d) Alcian blue and toluidine blue staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. Scale bars = 100 μ m. (e) Growth curves in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA, measured by CCK8-assay. (f) Relative Col2a1 and Sox9 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (g) Col2a1, Sox9, Cleaved Caspase-3, Bax, and Bcl-2 protein expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (h) Quantitative measurement of (g). (i) Relative Caspase-3 activity in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (j) TUNEL staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. White arrows indicate TUNEL-positive cells. Scale bars = 50 μ m. (k) Quantitative measurement of (j). (l) Relative IL-6 , COX , NOS2 , and MMP13 mRNA expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (m) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining, CCK-8 Assay, Activity Assay, TUNEL Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: si-CHOP reverses chondrocyte proliferation, apoptosis, and inflammatory response caused by USP7 knockdown under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (c) Quantitative measurement of (b). (d) Relative Col2a1 and Sox9 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (e) Col2a1, Sox9, Cleaved Caspase-3, Bax, Bcl-2, and PCNA protein expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (f) Quantitative measurement of (e). (g) Relative IL-6 , COX , NOS2 , and MMP1 3 mRNA expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (h) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: NF- κ B signaling inhibitor QNZ reverses chondrocyte proliferation, apoptosis, and inflammatory response caused by USP7 knockdown under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (c) Quantitative measurement of B. (d) Alcian blue and toluidine blue staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. Scale bars = 100 μ m. (e) Relative Col2a1 and Sox9 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (f) Col2a1, Sox9, Cleaved Caspase-3, Bax, Bcl-2, and PCNA protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (g) Quantitative measurement of F. (h) Relative Caspase-3 activity in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (i) TUNEL staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. White arrows indicated TUNEL-positive cells. Scale bars = 50 μ m. (j) Quantitative measurement of (i). (k) Relative IL-6 , COX , NOS2 , and MMP1 3 mRNA expression in USP7 knockdown and its control groups under TNF- α- induced inflammation after 48 h chondrogenic induction, with and without QNZ. (l) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining, Activity Assay, TUNEL Assay

Journal: Chinese Medical Journal

Article Title: Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

doi: 10.4103/0366-6999.190666

Figure Lengend Snippet: Collagen Type II alpha 1 IHC staining of the NP at each time point (original magnification, ×400). (a) Fresh tissue; (b, d, and f) tissue collagen Type II alpha 1 staining of organ controls at days 3, 7, and 14, respectively; (c, e, and g) collagen Type II alpha 1 staining of tissues under static load at days 3, 7, and 14, respectively. The intensity of staining remained constant by 14 days in the controls, and no significant difference was observed between the controls and fresh tissue (a, arrow). The samples under static load showed a significant enhancement in IHC intensity for the initial 3 days (c, arrowheads); however, the staining was obviously reduced after 3 days, by 14 days (g), the staining was significantly decreased (arrow) and significantly different from the controls. IHC: Immunohistochemistry; NP: Nucleus pulposus.

Article Snippet: Then, the sections were incubated with a mouse monoclonal primary antibody against collagen Type II alpha 1 (Sigma-Aldrich, Bale, Switzerland) diluted 1:20 overnight at 4°C.

Techniques: Immunohistochemistry, Staining

Journal: Chinese Medical Journal

Article Title: Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

doi: 10.4103/0366-6999.190666

Figure Lengend Snippet: Collagen Type II alpha 1 IHC staining intensity of the NP at each time point. No significant differences in collagen Type II alpha 1 staining intensity were observed between the fresh tissues and controls. The samples under static load showed a significant enhancement at 3 day, and significant differences were observed between the controls and fresh tissues. At 7 day, the staining was obviously decreased, and by 14 days, it was significantly decreased ( P < 0.05) and significantly different from that of the controls. The values represent the mean ± SD. * P < 0.05 versus day 0 of the same group; † P < 0.05 versus day 3 of the same group; ‡ P < 0.05 versus controls. IHC: Immunohistochemistry; NP: Nucleus pulposus; SD: Standard deviation.

Article Snippet: Then, the sections were incubated with a mouse monoclonal primary antibody against collagen Type II alpha 1 (Sigma-Aldrich, Bale, Switzerland) diluted 1:20 overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

doi: 10.4103/0366-6999.190666

Figure Lengend Snippet: Collagen Type II alpha 1 IHC staining intensity of the NP during organ culture

Article Snippet: Then, the sections were incubated with a mouse monoclonal primary antibody against collagen Type II alpha 1 (Sigma-Aldrich, Bale, Switzerland) diluted 1:20 overnight at 4°C.

Techniques: Immunohistochemistry

Journal: Chinese Medical Journal

Article Title: Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

doi: 10.4103/0366-6999.190666

Figure Lengend Snippet: Relative expression quantified by real-time PCR. An obvious downregulation of Agg was observed in the controls after 3 days whereas a significant downregulation was observed under static load during culture compared with fresh tissue and controls. An upregulation of COL2A1 was observed in response to static load by 3 days, and the expression of this gene subsequently decreased until day 14 to below detectable levels. COL2A1 was downregulated in the controls by 3 days whereas no significant downregulation was observed after culturing, and significant differences were observed between the two groups after culture. The values represent the means ± SD normalized to fresh tissue. * P < 0.05 versus day 0 of the same group; † P < 0.05 versus day 3 of the same group; ‡ P < 0.05 versus controls. PCR: Polymerase chain reaction; Agg : Aggrecan; COL2A1 : Collagen Type II alpha 1; SD: Standard deviation.

Article Snippet: Then, the sections were incubated with a mouse monoclonal primary antibody against collagen Type II alpha 1 (Sigma-Aldrich, Bale, Switzerland) diluted 1:20 overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation