Fig 2: HS3ST3B1-IT1 attenuates OA progression in vivo(A) Schematic diagram illustrating the experimental design. Mice were randomly assigned into three groups: Ctrl+AAV-NC group (n = 7), MIA+AAV-NC group (n = 7), and MIA+ AAV-IT1 group (n = 7). For the induction of OA, mice were given an intra-articular injection of MIA in the knee. One week later, AAVs were administered once per week for 3 consecutive weeks. Six weeks later, mice were euthanized, and the knee joint tissues were separated for the subsequent experiments.(B) Histological sections of cartilage harvested from each group were stained with safranin-O/fast green and toluidine blue staining. Scale bar, 100 µm (n = 4 for each group).(C) The Mankin scoring system was used to evaluate articular cartilage of mice from each group (n = 4).(D) The proliferation of chondrocytes in mouse cartilage tissues was detected using Ki67 immunohistochemistry. Upper panel: representative images of Ki67 staining (scale bar, 100 µm). Inset boxes indicate the areas of the articular cartilage. Lower panel: the percentage of Ki67-positive chondrocytes in mouse articular cartilage of each group (n = 4).(E) Apoptosis of chondrocytes in mouse cartilage tissue was evaluated using the TUNEL assay. Left panel: representative images of TUNEL staining (scale bar, 100 µm). Dotted lines indicate the area of the articular cartilages. Right panel: the quantification of TUNEL-positive cells in the articular cartilages from each group (n = 4).(F) The expressions of COL2A1 and MMP13 in knee articular cartilage were detected by IHC staining (scale bar, 100 µm). Inset boxes indicate the area of articular cartilage (n = 4 for each group).(G) The expression levels of HS3ST3B1-IT1 in the knee articular cartilage were detected by qRT-PCR. Data were presented as the mean ± SD (n = 3). Statistical differences were determined using an unpaired Student’s two-tailed t test (C–-E and G). Saf-O/FG, safranin-O/fast green; TB, toluidine blue staining; AAV, adeno-associated virus; IT1, HS3ST3B1-IT1; MIA, monosodium iodoacetate; NC, negative control; ns, no significant difference.

Fig 3: HS3ST3B1 increases chondrocyte viability, inhibits chondrocyte apoptosis, and regulates ECM metabolism(A and B) qRT-PCR (A) and western blotting (B) were performed to verify the overexpression and knockdown efficiencies of HS3ST3B1 in chondrocytes transfected with pCMV3-HS3ST3B1 plasmid or HS3ST3B1 siRNAs. Values were shown as mean ± SD (n = 3).(C) CCK-8 assay was used to identify the viability of chondrocytes with HS3ST3B1 overexpression or knockdown. Values were shown as mean ± SD (n = 3).(D) The apoptosis rates were evaluated in chondrocytes with HS3ST3B1 overexpression or knockdown by flow cytometry. Values were shown as mean ± SD (n = 3).(E) The expression levels of apoptosis-associated proteins were detected by western blotting in chondrocytes with HS3ST3B1 overexpression or knockdown. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel).(F) The expression levels of ECM proteins (COL2A1 and Aggrecan) and cartilage-degrading enzymes (MMP13 and ADAMTS-5) were analyzed by western blotting in chondrocytes with HS3ST3B1 overexpression or knockdown. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel). Statistical analysis was performed using an unpaired Student’s two-tailed t test (A, D–F) or a two-way ANOVA followed by Sidak’s multiple comparison test (C).

Fig 4: HS3ST3B1-IT1 increases chondrocyte viability, inhibits chondrocyte apoptosis, and regulates ECM metabolism(A) qRT-PCR was performed to verify the overexpression and knockdown efficiencies of HS3ST3B1-IT1 in chondrocytes transfected with pcDNA-HS3ST3B1-IT1 or HS3ST3B1-IT1 ASO. Values were shown as mean ± SD (n = 3).(B) CCK-8 assay was used to identify the viability of chondrocytes with HS3ST3B1-IT1 overexpression or knockdown. Values were shown as mean ± SD (n = 3).(C) Chondrocyte apoptosis was detected with FITC-Annexin V/PI double staining using flow cytometry following HS3ST3B1-IT1 overexpression or knockdown. Values were shown as mean ± SD (n = 3).(D) The expression levels of apoptosis-associated proteins were evaluated by western blotting. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel).(E) The expression levels of ECM proteins (COL2A1 and Aggrecan) and cartilage-degrading enzymes (MMP13 and ADAMTS-5) were analyzed by western blotting in chondrocytes following HS3ST3B1-IT1 overexpression or knockdown. Representative blots from three independent experiments were shown (left panel). Quantitative analyses of the blots were presented as mean ± SD (n = 3, right panel). Statistical analysis was performed using an unpaired Student’s two-tailed t test (A and C–E) or a two-way ANOVA followed by Sidak’s multiple comparison test (B).

Fig 5: Knockdown of HS3ST3B1 impairs the effects of HS3ST3B1-IT1 on chondrocyte viability, chondrocyte apoptosis, and ECM metabolism(A) Western blotting was performed to assess the protein levels of HS3ST3B1 in chondrocytes transfected with Vector+si-NC, Vector+si-HS3ST3B1, pcDNA-HS3ST3B1-IT1+si-NC, or pcDNA-HS3ST3B1-IT1+si-HS3ST3B1. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel).(B) CCK-8 assay was used to evaluate the viability of chondrocytes in each group. Values were shown as mean ± SD (n = 3).(C) The apoptosis rates were evaluated by flow cytometry in each group. Values were shown as mean ± SD (n = 3).(D) The expression levels of apoptosis-associated proteins were detected by western blotting. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel).(E) The expression levels of ECM proteins (COL2A1 and Aggrecan) and cartilage-degrading enzymes (MMP13 and ADAMTS-5) were analyzed by western blotting in each group. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel). Statistical differences were determined using an unpaired Student’s two-tailed t test (A and C–E) or a two-way ANOVA followed by Tukey’s multiple comparison test (B).