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. 2022 Mar 5;23(5):2864. doi: 10.3390/ijms23052864

Figure 6.

Figure 6

Activation status of TGF-β1 signaling pathways on chondrocyte. (A) Signaling events induced by TGF-β1. Rat chondrocytes were untreated or exposed to 100 µg/mL of nanoliposomes encapsulating or not encapsulating TGF-β1 (1 ng/mL), or to TGF-β1 alone (10 ng/mL) for 10 min (p-ERK and p-p38-MAPK) or 3 h (p-Smad3/5). Total proteins were extracted from cells and subjected to Western blotting using anti-phospho-ERK, anti-phospho-p38-MAPK or anti-phospho-Smad3/5. The relative abundance of these proteins was normalized to that of β-actin protein. The reported data are represented as mean ± SD of at least three individual experiments. Significance is indicated as * when compared to Ctrl and # when compared to TGF-β1 alone (p < 0.01). (B) Rat chondrocytes were exposed to 100 µg/mL of nanoliposomes encapsulating or not encapsulating TGF-β1 (1 ng/mL) for 24 h (mRNA) or 48 h (PPi), and with or without 10 μM of SB203580 (a selective p38-MAPK inhibitor) or 30 μM of PD98059 (a MEK-1 inhibitor) added 1 h before TGF-β1. Total RNA was extracted then reverse transcribed into cDNA and analyzed by real-time PCR. The relative abundance of Col 2 and Ank was normalized to RP29. Quantifications were made by using the ΔΔCt method. ePPi levels in culture supernatant of rat chondrocytes were assayed radiometrically and normalized to the amount of total cell proteins. The reported data are represented as mean ± SD of at least three (mRNA) or six (ePPi) individual experiments. Significance is indicated as * when compared to NL and # (p < 0.01). Ank—inorganic pyrophosphate transport regulator; Ctrl—control cells, meaning untreated chondrocytes; ePPi—extracellular inorganic pyrophosphate; ERK, extracellular signal-regulated kinase; MEK-1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1; NL—empty nanoliposomes; NL-TGF—TGF-β1-loaded nanoliposomes; NL-TGF 1—nanoliposomes encapsulating 1 ng/mL of TGF-β1; NL-TGF 1-ERK—condition NL-TGF 1 after ERK pathway inhibition (PD98059); NL-TGF 1-p38—condition NL-TGF 1 after p38 pathway inhibition (SB203580); p38-MAPK—p38 mitogen-activated protein kinase; PCR—polymerase chain reaction; p-ERK—phosphorylated ERK or phospho-ERK; p-P38—phosphorylated p38 or phospho-P38; p-Smad3/5—phosphorylated pSmad3/5 or phospho- pSmad3/5; RP29—ribosomal protein 29; SD—standard deviation; TGF-β1—transforming growth factor-β1; TGF 10—TGF-β1 alone at a concentration of 10 ng/mL.