Morphological alterations in PKCι depleted cells expressing Htt-103.
A) U87MG cells transfected with the control or with siPKCιA and siPKCιB were transiently transfected with plasmids encoding GFP, Htt-25 or Htt-103 for 24 hours. Cell morphology was assessed by phase contrast microscopy. An increase in the number of shrunken, rounded and detached cells was observed in PKCι depleted cells expressing Htt-103 when compared to control RNA transfected cells or cells expressing GFP or Htt-25. Magnification was 40×. B) U87MG cells expressing GFP, Htt-25 or Htt-103 for 24 hours were visualized under fluorescence (bottom panel) to assess transfection efficiency. Upper panels represent the same field of view visualized under white light. Arrowheads demonstrate nuclear inclusions in Htt-103 expressing cells. Scale bars represent 100 µm. C) Western blot analysis with a PKCι specific antibody of cell extracts from U87MG cells transfected in duplicate with either the control or siPKCιA and siPKCιB showing the reduction in the protein levels of PKCι at 24 and 48 hours post-transfection. The membrane was re-probed with an antibody directed against Pan-ERK which served as a loading control. D) Western blot analysis of cell extracts from cells transfected with either control or siPKCιA expressing GFP, Htt-25 or Htt-103 with an antibody raised against AFP. No significant difference in the protein levels of GFP, Htt-25 and Htt-103 were observed in extracts from control and PKCι transfected cells. The membrane was re-probed with an antibody directed against Pan-ERK which served as a loading control.