Fig. 3

Partially overlapping subcellular localization of PI3K-C2α and PI3K-C2β in HUVECs. a Cells were cotransfected with the expression vectors of green fluorescent protein (GFP)-C2α and mCherry-C2β and imaged by confocal microscopy coupled with super-resolution radial fluctuation (SRRF)-Stream mode. Nuclei were stained with DAPI (blue). Note the cytoplasmic fine puncta of GFP-C2α (left) and coarse and fine puncta of mCherry-C2β (middle) surrounding the podosomes and on the plasma membrane (white arrowheads) and perinuclear coarse puncta of mCherry-C2β (yellow arrows). White arrowheads in the SRRF view of the merged image indicate the co-localization of GFP-C2α and mCherry-C2β. b Representative confocal microscopic images of GFP-C2α or mCherry-C2β and F-actin. Cells transfected with the expression vectors of GFP-C2α and mCherry-C2β were stained with iFluor 488-conjugated phalloidin (green) or Alexa Fluor 594-conjugated phalloidin (red). Nuclei were stained with DAPI. White arrowheads indicate the co-localization of F-actin and GFP-C2α or mCherry-C2β. Blue arrowheads indicate the co-localization of F-actin and mCherry-C2β in the membrane ruffle. c, d Co-localization of GFP-C2α (c) or mCherry-C2β (d) with organelle markers. Cells expressing either GFP-C2α or mCherry-C2β were stained with antibodies against CHC (clathrin-coated structures [CCS]), EEA1 (early endosomes [EE]), Rab7 (late endosomes [LE]), LAMP1 (lysosomes [LY]) and LC3B (autophagosomes [AP]). Nuclei were stained with DAPI (blue). White arrowheads in SRRF views indicate the co-localization of GFP-C2α or mCherry-C2β with each organelle marker