Figure 4.

Phospho-mimetic isoform, Cx37-S275D, triggers cell death in a density-dependent manner. (A) Representative MS/MS spectra identifying phosphorylation at serine 275 in the mid-tail region of Cx37. Assignment of the indicated ion fragments is shown. Although they show that other possible phosphorylation targets were not phosphorylated, they do not discriminate between S274 vs. S275 as the phosphorylated residue. (B) At low density, expression of Cx37-S275D (orange) results in cell death – see in particular the inset. But when expression was induced at higher cell density, where cell-cell contact occurred, no cell death was evident. n = 4 for WT, n = 3 for S275D for each dox condition. dox -, no protein expression; dox +, induced Cx37 expression; Δ dox, non-expressing iRin37-S275D cells grew to a higher cell density for 14 days, after which dox was included in the media to induce expression, denoted by grey hash. p < 0.05 for dox + versus dox − for -S275D (†), as well as -WT (∗). For the dox switch experiment, growth rate before versus after addition of dox was not different. All values are mean ± s.e.m (where error bars are not evident, they are smaller than the symbol size). (C) As assessed by propidium iodide DNA staining and FACS, an increase in the number of initially plated cells per 100 mm dish (indicated at the top of each FACS graph), and therefore cell density, reduced the extent of Cx37-S275D-induced cell death. % dead cells in 10⁴ analyzed events: 0.5 × 10⁶ cells, 88%; 3 × 10⁶ cells, 72%; 4 × 10⁶ cells, 55%. Arrowheads on x axis denote the G₀/G₁ peak center as identified by the Modfit LT program when possible. Otherwise, peak centers were user defined.