1. Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis 
E.W. Bradley, L.R. Carpio, M.E. McGee-Lawrence, C. Castillejo Becerra, D.F. Amanatullah, L.E. Ta, M. Otero, M.B. Goldring, S. Kakar, J.J. Westendorf 
Osteoarthritis and Cartilage 
Volume 24, Issue 6, Pages (June 2016)
DOI: /j.joca
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Phlpp1 deletion protects against post-traumatic osteoarthritis. Twelve-week-old male WT (n = 5) or Phlpp1−/− (n = 7) mice were subjected to DMM surgeries and evaluated 12 weeks post-surgery. (A) Safranin O/Fast Green staining from WT and Phlpp1−/− mice subjected to DMM surgeries. The left panels are 4× magnifications of the knee joint and the right panels are 40× magnifications of the medial knee surfaces. (B) OARSI scores from WT and Phlpp1−/− mice, *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Phlpp1 deletion minimizes subchondral bone thickening associated with post-traumatic OA. (A) Radiographs from WT and Phlpp1−/− mice 12 weeks-post DMM surgery. The arrow denotes an area of increased subchondral thickness. (B) Mean gray values of the medial subchondral bone radiographs shown in (A) from WT (n = 5) and Phlpp1−/− (n = 7) mice were determined using Image J software, *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Pain following OA surgery is attenuated by Phlpp1 deficiency. Percent change in paw withdrawal frequency from baseline using a 0.16 g filament observed at 6 and 12 weeks post-surgery, *P < 0.05, #P < 0.1.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Phlpp1 deficiency increases cellular content and articular thickness. (A) Sections from paraffin embedded tibiae of 4-week-old WT (n = 3) or Phlpp1−/− (n = 4) mice were stained with Alcian blue and (B) the number of articular chondrocytes per tissue area was determined, *P < 0.05. (C) Articular cartilage thickness (mm) of 4-week-old WT (n = 7) and Phlpp1−/− (n = 7) mice was measured by EPIC μCT, #P = 0.07.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
PHLPP1 expression is elevated in human OA tissue. Surgically discarded tissue from total joint arthroplasties due to OA or FNFx repair was collected. (A) Sections from paraffin embedded tissues were incubated with an isotype control IgG (left column) or an anti-PHLPP1 antibody (right columns). Shown are representative 10× and 40× images. (B) Average PHLPP1 staining intensity from OA (n = 6) and FNFx (n = 4) tissues was determined using Image J software, *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
The human PHLPP1 promoter is demethylated in OA. (A) Genomic DNA was isolated from FNFx articular cartilage (n = 9). Damaged and undamaged portions of articular cartilage from total knee arthroplasty specimens (n = 5) were also collected. Percent methylation of the CpG island within the Phlpp1 promoter using the Epitect II DNA Methylation kit for each sample was determined. (B, C) Genomic DNA was isolated from FNFx (n = 6) or OA (n = 14) specimens and RRBS was performed. (B) Differentially methylated CpG sites and putative transcription factor binding sites in the PHLPP1 promoter. Percent methylation at each site for each specimen (FnFx: F, OA: O) is noted by shading intensity. (C) Average percent methylation at each CpG site was determined in all OA or FNFx specimens from (B), *P < 0.05, #P < 0.06. (D) C28/I2 and T/C28a2 human chondrocytes were treated with 5-azacytidine (AzaC) or PBS. Expression of PHLPP1 was determined by qPCR, *P < 0.05. (E) The PHLPP1-LUC reporter or empty vector were treated in vitro with the CpG methyltrasferase M. Sssl or left untreated. Plasmids were then transfected into T/C28a2 cells and luciferase activity was measured. (F) T/C28a2 chondrocytes were treated with the indicated inflammatory mediators for 1 h and expression of PHLPP1 was evaluated by qPCR, *P < 0.05. The dotted line denotes baseline levels (1-fold).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions