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. 2019 Oct 25;15(10):e1008096. doi: 10.1371/journal.pgen.1008096

Fig 5. Adgrg6 regulates gene expression profiles and STAT3 signaling in ATDC5 cell culture.

Fig 5

(A) Schematic of a 17-bp deletion of Adgrg6 from a stable single cell clone of ATDC5 cell line (Adgrg6 KO). (B) qPCR analyses of gene expression in ATDC5 cells at 5 and 15 days of maturation demonstrates decreased expression of markers of healthy disc, Col2a1and Sox9, and increased expression of the hypertrophic marker, Col10a1 and the extracellular matrix modifying enzyme, Mmp13 in Adgrg6 KO cells. (C) qPCR analyses revealed increased expression of Socs3 in Adrgr6 KO cells after 15 days of maturation. (D) qPCR analysis of Il1a, Il1b, Il6 and Tnf in ATDC5 cells maturated for 15 days. (E) qPCR analysis of Il6 expression in 1.5-month-old primary mouse IVDs (induced from E0.5-P20). (B-E, n = 3 biological replicates and representative result is shown. Bars represent mean ± SD. *p≤0.05, **p≤0.01, two-tailed Student's t Test). (F) Representative Western blot of wild type and Adgrg6 KO ATDC5 cell lysates showing stimulation of pSTAT3 staining after treatment with recombinant IL-6 (rIL-6) protein in both cell lines, while Adgrg6 KO cells show a mild constitutive stimulation of pSTAT3 without addition of rIL-6 (n = 4 biological replicates and representative result is shown). (G) Densitometry of the Western blot of wild type and Adgrg6 KO ATDC5 cell with or without IL-6 (rIL-6) treatment. (Each dot represents one biological replicate. Bars plot with mean ± SD. *p≤0.05, two-tailed Student's t Test.)