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JCB_22554_sm_SupplFigS1.tif215KSupplementry Figure S1. Semi-quantitative RT-PCR performed on LN18 cell line for Cx26 (forward: 5′ TGG CCT ACC GGA GAC AT 3′; reverse: 5′ CGT CGT ACA TGA CAT AGA AGA C 3′), Cx30 (forward: 5′ TTC CTT TAC AAT GGG TAC CA 3′; reverse: 5′ TTA AGC AGC ATG CAA ATC CA 3′), Cx32 (forward: 5′ TCC CTG CAG CTC ATC CTA GT 3′; reverse: 5′ CCC TGA GAT GTG GAC CTT GT 3′), Cx40 (forward: 5′ TAG GCA AGG TCT GGC TCA CT 3′; reverse: 5′ TGA TCT GCA GCA CCC AGT AG 3′), Cx43 (forward: 5′ CGG TTG AGT CAG CCT GG 3′; reverse: 5′ TCT CTT CCT TTC GCA TCA CAT A 3′), Cx45 (forward: 5′ CCA ACC AAA ACC TAA GCA TG 3′; reverse: 5′ ACA TAA AAC GGG TGG ACT TG 3′) and Cx46 (forward: 5′ GCC GGC CAG TAC TTT CTG TA 3′; reverse: (5′ CCT GCT TGA GCT TCT TCC AG 3′). Following the reverse transcription of mRNA to cDNA, 28 PCR cycles were performed (92°C, 1 min; 58°C, 1 min; 72°C, 1 min). Among the amplified cDNA, Cx43, Cx45 and Cx46 were detected. C: control genomic DNA from human HeLa cells; +: cDNA from RT-PCR performed on LN18 cell line. Negative controls, without RT, were loaded and did not exhibit genomic DNA contamination (not shown). GAPDH was used as a quality control for cDNA.
JCB_22554_sm_SupplFigS2.tif13598KSupplementry Figure S2. U87 and U251 glioma cell lines transduced by different constructs of Cx43. A: Detection of Cx43, TrCx43 and 243Cx43 by western blotting. The different forms were detected at the expected size at 43 kDa, 30 kDa and 15 kDa respectively. GAPDH was used as a loading control. B: Communication assay on U87 and U251 cell lines expressing Cx43, TrCx43 or 243Cx43. Donor cells were preloaded with DiI and Calcein-AM and trypsinized. An aliquot of these cells was seeded on a confluent culture of receiving cells with the same phenotype. After 4 hours, diffusion of Calcein-AM via the GJIC was observed. DiI, unable to diffuse via GJIC, marked the donor cells. The number of coupled cells was estimated by counting the number of cells stained by Calcein-AM. Only the full-length Cx43 appeared to be able to restore GJIC. Student t-test: ***, p<0.001. C: Anchorage-independent growth was evaluated by soft agar assay for U87 and U251 cell lines expressing Cx43, TrCx43 or 243Cx43 were plated at 10,000 cells in soft agar solution 0.3% in DMEM complemented by 10% fetal calf serum. Colonies′ size was estimated after 2 weeks in 30 randomized fields. Every constructs, Cx43, TrCx43 and 243Cx43, induced a decreased ability to grow in soft agar. Student t-test: **, p<0.01; ***, p<0,001. D: Migration ability was monitored by transwell assay or by wound healing assay. For U87 cell line, the transwell assay was chosen because of the particular cell morphology and migration behavior. U87 cells had elongated morphology and tended to migrate independently of each other, making difficult to determine the area of migration. Cells (0.1x106) were seeded in triplicates at both the top of a transwell insert (BD Biocoat 8.0-µm inserts in 24-well plates, BD Biosciences) and in a separate well without the transwell insert. Cells were incubated for 16 hrs and subsequently trypsinized from the bottom of the insert (number of traversed cells) and the separately seeded well (number of total cells) for cell counting using Z1 Coulter Particle Counter with IsoFlow Sheath Fluid as a diluent. Cell motility was determined as number of traversed cells per number of total cells. Every constructs Cx43, TrCx43 and 243Cx43, induced an increased migration. Student t-test: **, p<0.01; *, p<0.05. For U251 cell line, a wound healing assay was performed as well as a transwell assay (not shown). Twenty-four hours after a wound, the covered distance was estimated in 8 different areas of the culture. In both assays, the expression of Cx43 full-length or truncated forms of the protein did not have any effect on cell migration.

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