Fig. 2: TM4SF5 dynamically induced CD44v8-10 protein complexes with CD98hc and xCT under ROS-generating conditions.

a, b Cells transduced with control Strep (−) or Strep-TM4SF5 plasmid-containing retrovirus were transfected with FLAG-CD44v8-10 plasmid before preparation of whole cell lysate for pull-down with streptavidin-agarose beads and immunoblot analysis. c A schematic showing the quantity of molecules binding to TM4SF5 or CD44 during proteomic analysis. d, e Cells transduced without (d) or with (e) HA-xCT-encoding retrovirus were transfected with control Strep or Strep-TM4SF5 plasmid. Whole-cell lysates were pulled-down with streptavidin-agarose beads for western blot analysis. f, g Cells stably transduced with control (−) or Strep-TM4SF5-encoding retrovirus were treated with control vehicle (−) or TNF-α for the indicated times. Whole cell lysates were processed for co-precipitation analysis. h Cells transduced with control (−) or HA-TM4SF5 retrovirus were kept in different serum concentrations (0, 5, or 10%) before measurement of DCF-DA fluorescence to stain for intracellular ROS. i, j Cells transduced with control (−) or HA-TM4SF5 retrovirus were kept in different serum concentrations without (i) or with NAC treatment (j). Whole-cell lysates were processed for co-precipitation analysis. k Cells transfected without (−) or with Strep-TM4SF5 ( + ) were treated with vehicle DMSO (−) or TSAHC for 2 h. Whole-cell lysates were processed for co-precipitation analysis. l Cells were transfected with control (−) or Strep-TM4SF5 ( + ) together with wild-type (WT) or S301A mutant FLAG-CD44v8-10 plasmids, prior to TNF-α treatment (2 ng/ml) for 6 h. Whole-cell lysates were processed for co-precipitation analysis. P values were calculated by two-tailed, unpaired Student’s t-test. P values <0.05 were considered statistically significant. Data represent three isolated experiments. See also Figure S2