Research Paper Volume 13, Issue 8 pp 11433—11454

Overexpression of long non-coding RNA AP001505.9 inhibits human hyaline chondrocyte dedifferentiation

Transfection of AP001505.9 overexpression lentivirus. P5 human chondrocytes were inoculated into a 24-well plate at a density of 5 × 104/well. After 24 h, culture medium was replaced with viral transfection solution. Cells were transduced with AP001505.9 overexpression viruses with a multiplicity of infection (MOI) = 75. (A) Phase contrast microscopy and fluorescence microscopy; green fluorescence represents successful chondrocyte transfection. LNC represents chondrocytes transfected by AP001505.9 lentivirus. NC represents chondrocytes transfected by negative control lentivirus. (B) Real-time qPCR for AP001505.9. (C) Real-time qPCR for COL1A1, COL2A1, and SOX-9. (D, E) Western blotting analysis of COL1, COL2, and SOX-9 levels. (F–H) Immunofluorescence staining of COL1, COL2, and SOX-9; NC and LNC chondrocytes are stained with anti-COL2, anti-COL1, anti-SOX-9, and Hoechst 33342. GAPDH was used as the internal reference. Data are represented as means ± standard deviation. *P P P

Figure 5. Transfection of AP001505.9 overexpression lentivirus. P5 human chondrocytes were inoculated into a 24-well plate at a density of 5 × 104/well. After 24 h, culture medium was replaced with viral transfection solution. Cells were transduced with AP001505.9 overexpression viruses with a multiplicity of infection (MOI) = 75. (A) Phase contrast microscopy and fluorescence microscopy; green fluorescence represents successful chondrocyte transfection. LNC represents chondrocytes transfected by AP001505.9 lentivirus. NC represents chondrocytes transfected by negative control lentivirus. (B) Real-time qPCR for AP001505.9. (C) Real-time qPCR for COL1A1, COL2A1, and SOX-9. (D, E) Western blotting analysis of COL1, COL2, and SOX-9 levels. (FH) Immunofluorescence staining of COL1, COL2, and SOX-9; NC and LNC chondrocytes are stained with anti-COL2, anti-COL1, anti-SOX-9, and Hoechst 33342. GAPDH was used as the internal reference. Data are represented as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Scale bar, 100 μm.