primary antibodies against collagen type ii alpha 1 chain  (Huabio 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