1. PKCε is a regulator of hypertrophic differentiation of chondrocytes in osteoarthritis 
V. Queirolo, D. Galli, E. Masselli, R.M. Borzì, S. Martini, F. Vitale, G. Gobbi, C. Carubbi, P. Mirandola 
Osteoarthritis and Cartilage 
Volume 24, Issue 8, Pages (August 2016)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
(A) Representative immunoblotting for PKCε in OA chondrocytes collected at day 0 of culture (0) and after 1 week of culture7 in micromass or monolayer. Actin was monitored for protein loading. (B) Densitometry analysis of PKCε signal was performed on three separate experiments from three patients using Image J Software. Reported values have been normalized to actin (means ± SD, *P < 0.05 vs T0, Dunnett tests).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
(A) Toluidine Blue on 4 μm thick slices of paraffin embedded, ShRNA infected cell grown 7 day in micromass culture. Representative images of one patient. Acidophilic metachromatic dye complexes with anionic glycoconjugates as PG and GAG. (B) Alizarin Red dye, representative images from two separate experiments. The dye specifically binds to calcium deposits, resulting in red blots, indicators of mineralization processes, as shown in the enlarged inserts (a, b, c).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
(A, B) Levels of COL10 and COL2 mRNA, respectively, in monolayer culture of OA chondrocytes from three patients, collected 7 days after infection with lentiviral vector containing PKCε specific ShRNA (ShRNAε) or ShRNA control (ShRNA CT). Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments (*P < 0.01 vs control, Student's t-test). (C) Representative immunofluorescence of COL2 performed on OCT-frozen micromasses obtained after ShRNA treatment and collected at day 7 of culture. Slices were labeled with specific antibody against COL2 and detected with anti-rabbit conjugated to an Alexa Fluor 488. Original magnification is reported; 50 μm bar is indicated.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
(A) levels of SOX9 mRNA in monolayer culture of OA chondrocytes from three patients, collected at day 7 after infection with lentiviral vector containing PKCε specific ShRNA (ShRNAε) or ShRNA control (ShRNA CT). Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments (*P < 0.00 vs control, Student's t-test). (B) Representative Immunoblotting for PKCε and SOX9 on OA chondrocytes collected after 7 days from PKCε specific ShRNA (ShRNAε), or ShRNA control (ShRNA CT) lentiviral infection. Uninfected cell cultures are also reported (UNTR). Actin was monitored for protein loading. The graph shows SOX9 densitometry analysis: the expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments (*P < 0.00 vs control, Student's t-test). (C–D) Representative immunofluorescence of SOX9 performed on OCT-frozen micromasses obtained after ShRNA treatment and collected at day 7 of culture. Slices were labeled with specific antibody against SOX9 and detected with anti-rabbit conjugated to an Alexa Fluor 488; Tubulin α and Tubuline β antibodies were detected with specific anti-mouse Alexa Fluor 546. White arrows indicate localization of SOX9 in perinuclear regions in the PKCε-KD sample. Original magnification is reported; 10 μm and 100 μm bars are indicated.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
(A)Levels of MMP13 mRNA in monolayer culture of OA chondrocytes from three patients, collected 7 days after infection with lentiviral vector. Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments (*P < 0.05 vs control, Student's t-test). (B)Secreted MMP-13 was detected by ELISA assay in culture supernatants. Concentration values were normalized to control cultures (**P < 0.01 vs control, Student's t-test). (C) Levels of HDAC1 mRNA in monolayer culture of OA chondrocytes from three patients, collected at day 7 after infection with lentiviral vector containing ShRNAε or shRNA CT. Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments. (D) Levels of HDAC3 mRNA in monolayer culture of OA chondrocytes from three patients, collected at day 7 after infection with lentiviral vector containing ShRNAε or shRNA CT. Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments. (E) Levels of HDAC5 mRNA in monolayer culture of OA chondrocytes from three patients, collected at day 7 after infection with lentiviral vector containing ShRNAε or shRNA CT. Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments. (F) Levels of HDAC2 mRNA in monolayer culture of OA chondrocytes from three patients, collected at day 7 after infection with lentiviral vector containing ShRNAε or shRNA CT. Gene expression, normalized to the control cultures (ShRNA CT), is reported as mean ± SD of three separate experiments (***P < 0.001 vs control, Student's t-test). (G) Levels of HDAC4 mRNA in monolayer culture of OA chondrocytes from three patients, collected at day 7 after infection with ShRNAε or shRNA CT. (*P < 0.05 vs control, Student's t-test). (H) Secreted MMP-13 was detected by ELISA assay in cells supernatants of three cultures obtained from three patients after a week of treatment with HDACs inhibitors. Concentration values were expressed as fold change in respect of the control after normalization for the untreated (*P < 0.05 vs untreated, Dunnett tests). (I) Representative Immunoblotting for PKCε and RUNX2 on OA chondrocytes collected after 7 days from the lentiviral infection. Protein expression of PKCε specific ShRNA (ShRNAε), or ShRNA control (ShRNA CT) lentiviral infection cultures is reported. Uninfected cell cultures are also shown (UNTR). Actin was monitored for equal protein loading. (L) RUNX2 densitometry analysis: the OD value of each ShRNA PKCε treatment was normalized for the corresponding control (ShRNA CT) (*P < 0.05 vs control, Student's t-test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
A schematic representation of the observed correlation between PKCε expression levels and OA hypertrophic phenotype in chondrocytes.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions