Biochemical evidence for gap junctions and Cx43 expression in immortalized human chondrocyte cell line: a potential model in the study of cell communication in human chondrocytes  R. Gago-Fuentes, P. Carpintero-Fernandez, M.B. Goldring, P.R. Brink, M.D. Mayan, F.J. Blanco  Osteoarthritis and Cartilage  Volume 22, Issue 4, Pages 586-590 (April 2014) DOI: 10.1016/j.joca.2014.02.002 Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 T/C-28a2 cells contain functional Cx43 channels. (A) Expression analyses of Cx43 mRNA by real-time RT-PCR in PC isolated from the cartilage (PC) of adult donors and T/C-28a2 (T/C) cells. Data were normalized to HPRT1 levels. Data are represented as the mean ± S.D. with n = 5. **P = 0.0043; Mann–Whitney test. (B) Western blots of T/C-28a2 cells from two confluent monolayer cultures. α-tubulin was used as loading control. IHC using the monoclonal anti-Cx43 antibody and cells on monolayer (left) and pellet (right) cultures after 7 days of culture in micromass. (C) Both cell types showed LY uptake consistent with hemichannels behaviour. The percentage of LY positive cells was calculated from six fields of view from two independent experiments (mean ± S.D.). (D) Voltage-gating properties of T/C-28a2 cells. (E) Dye (NBDG) flux in T/C-28a2 cell pairs. A pipette containing the dye was attached to the cell on the right in the whole cell configuration. Epifluorescent micrographs were taken at 0, 10, and 17 min after dye injection into the right-sided cell. Osteoarthritis and Cartilage 2014 22, 586-590DOI: (10.1016/j.joca.2014.02.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 GJIC between cells. (A) In situ electroporation on a partly conductive slide for the measurement of the intercellular junction transfer of LY. Cells were electroporated in the presence of 5 mg/ml of LY. After washing off the unincorporated LY, the cells were photographed under phase contrast or fluorescence illumination. The solid red lines define the conductive (on the left) and nonconductive side points (on the right). Original magnifications ×10 and ×20. The graph represents the number of contacted cells into which the dye was transferred per electroporated border cell (means ± S.D). n = 6 **P < 0.01, ***P = 0.0002; Mann–Whitney test, untreated (PC) vs GAP27 and PC vs T/C-28a2. (B) Scrape load/dye transfer assay was performed to examine GJ activity. The distance of dye transfer from cutting edge (solid vertical red line and green stars) to the farthest cells with the dye uptake is represented by horizontal red arrows or yellow stars. (C) Western blot assays to detect the amount of Cx43 and c-Src present in PC and T/C cells. α-tubulin was used as loading control. (D) IHC of T/C-28a2 cells and PC using anti-c-Src antibody. The negative controls are shown on the right. Osteoarthritis and Cartilage 2014 22, 586-590DOI: (10.1016/j.joca.2014.02.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. S1 (A) LY uptake for PC and T/C cells. No significant differences were found between untreated and treated cells with GAP27 for 1 h. (B) Expression analyses of HPRT1, pannexins and Cx43 mRNA by real-time RT-PCR in PC and T/C cells. Data are represented as the mean ± S.D. with n = 2. Primers used were specific for PANX1 5′-CAGAGCGAGTCTGGAAACCT-3′ and 5′-GCAGGCTCCATCTCTCATGT-3′ and for PANX2 5′-TGCTGGTCACCCTGGTCT-3′ and 5′-GCGTAGGGCAGGAACTTGT-3′. Osteoarthritis and Cartilage 2014 22, 586-590DOI: (10.1016/j.joca.2014.02.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. S2 (A) Expression analyses of Cx43 mRNA by real-time RT-PCR in primary chondrocytes isolated (PC), T/C-28a2 (T/C) cells and human adult articular cartilage. Data were normalized to HPRT1 levels. Data are represented as the mean ± S.D. with n = 7. (B) IHC using the monoclonal anti-Cx43 antibody and PC, T/C and normal adult human cartilage. (C) Expression analyses of miRNA1-1 by real-time RT-PCR in PC, T/C cells and cartilage. Data were normalized to HPRT1 levels and are represented as the mean ± S.D. with n = 7. Osteoarthritis and Cartilage 2014 22, 586-590DOI: (10.1016/j.joca.2014.02.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions