1. Lentiviral vector-mediated shRNAs targeting a functional isoform of the leptin receptor (Ob-Rb) inhibit cartilage degeneration in a rat model of osteoarthritis 
S.L. Deng, J. Zhu, Q. Huang, W.L. Fu, Q. Li, G. Chen, X. Tang, J. Li 
Osteoarthritis and Cartilage 
Volume 25, Issue 11, Pages (November 2017)
DOI: /j.joca
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
(A) DNA sequencing confirmed that the shRNA3 sequences had inserted correctly into the pGC-LV shuttle plasmids. (B) Optimization of the transfection conditions. Representative examples of flow cytometry and confocal microscopy measurements showing that the transfection efficiency is 86.41% under the optimal transfection condition (Enhanced Infection Solution with 30 multiplicity of infection (MOI) lentiviral vectors). (C) Selection of optimal LV-shRNA. qRT-PCR (left) and western blotting (right) analysis showed that LV-shRNA3 achieved the highest silencing efficiency, as its efficiency was greater than that of the LV-NTCs; therefore, LV-shRNA3 was used for the subsequent experiment. Significant differences are indicated. RQ, relative quantity.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
In vitro Ob-Rb downregulation influenced the mRNA expression levels of pro-inflammatory (TNF-α, IL-1β and IL-6), anabolic (type II collagen) and catabolic (ADAMTS-4, ADAMTS-5, MMP-9, COX-2 and NOS-2) mediators. Relative expression levels (means with 95% confidence intervals (CIs)) in the LV-shRNA3 and Normal groups compared to the LV-NTC group were calculated using the comparative CT (2-△△CT) method. Significant differences are indicated. RQ, relative quantity.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Representative western blotting bands for ADAMTS-5, MMP-9 and type II collagen. These results were consistent with the corresponding qRT-PCR results indicated in Fig. 2.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Comparisons of cartilage histological findings and Osteoarthritis Research Society International (OARSI) scores among the groups at 2, 4, 8 and 12 weeks post-transfection. Sham, sham operation with PBS injection; OA, osteoarthritis model with PBS injection; OA + LV-NTC, osteoarthritis model with lentiviral vector-mediated non-targeting control sequence injection; OA + LV-shRNA3, osteoarthritis model with lentiviral vector-mediated Ob-Rb shRNA3 injection. (A) Representative sections stained with toluidine blue are shown at 5× magnification. ac – articular cartilage; sd – surface discontinuity; er – erosion; den – denudation; def – deformation. Scale bar, 200 μm. (B) Comparison of OARSI scores among the groups at 2, 4, 8 and 12 weeks post-transfection. Data are presented as means with 95% CIs. *P ≤ 0.001 vs OA; #P < 0.05 vs OA.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Comparison of immunohistochemical staining and semi-quantitative analysis results among the groups at 4 weeks post-transfection. Sham, sham operation with PBS injection; OA, osteoarthritis model with PBS injection; OA + LV-NTC, osteoarthritis model with lentiviral vector-mediated non-targeting control sequence injection; OA + LV-shRNA3, osteoarthritis model with lentiviral vector-mediated Ob-Rb shRNA3 injection. (A) Representative sections subjected to immunohistochemical staining for Ob-Rb and ADAMTS-5, MMP-9 and type II collagen expression (×400). Immunolabeled chondrocytes (arrow). (B) Semi-quantitative analysis. The proportions of chondrocytes that stained positive for Ob-Rb, ADAMTS-5 and MMP-9 were calculated and expressed as the percentages of positively stained cells within each region. Data are presented as means with 95% CIs. Significant differences vs OA group are indicated.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions