Integrin-α9 versus integrin-β1 expression. A. Integrin-α9, integrin-β1 and actin expression across eight glioma cell lines by reverse transcription-polymerase chain reaction (RT-PCR) analysis. B. Quantitative RT-PCR analysis of integrin-α9 versus integrin-β1 expression in glioma cell lines Hs683, U87 and U373. C. Hs683 expression levels of integrin-α9 and integrin-β1 under control (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) conditions obtained through microarray analysis using the Affymetrix Human Genome U133 set Plus 2.0.

Integrin trafficking and endoplasmic reticulum (ER) to Golgi protein export pathways.A. Integrin trafficking depicting the two main pathways of internalization, clathrin-mediated and caveolar, as well as the pathways for recycling. Clathrin-mediated internalization utilizes clathrin coated pits, whereas caveolar internalization utilizes dynamin GTPase to form vesicles containing lipid rafts and caveolin protein. Recycling occurs through fusion to early endosomes and proceeds either via Rab4 signaling through a short loop pathway or via Rab11 signaling through a long loop pathway where proteins pass through the perinuclear recyling complex. Respective integrins and their trafficking mediators are listed above the suggested pathways that they utilize (9, 17, 19, 30, 31). B. ER to Golgi protein export depicting coat protein complex II (COPII)-mediated anterograde transport and coat protein complex I (COPI)-mediated retrograde transport between the ER and the Golgi complex. COPII assembly commences with Sar1 G-protein activation by the Sec12 guanine nucleotide exchange factor, followed by the recruitment of the Sec23–24 heterodimer that recognizes ER export signals on cargo proteins for inclusion in vesicles. The Sar1-Sec23–24 complex recruits the Sec13–31 heterodimer that facilitates final budding and vesicle formation from the ER. Vesicles proceed to the ER–Golgi intermediate compartment (ERGIC), which is also called vesicular tubular clusters (VTC), the complex of which transports proteins to the Golgi destination for eventual final export (15, 20, 23). GTP = guanosine-5′-triphosphate; SNARE = N-ethylmaleimide-sensitive fusion protein attachment protein receptor. PKC = protein kinase C.

Transient transfection of galectin-1 using targeted small interfering RNA (siRNA) decreases intracellular galectin-1 protein. WB analysis of galectin-1 expression levels at days 5, 7 and 9 post-transfection in control (ctrl), scramble-transfected (scr), and galectin-1-transfected (siGal1) U87 (A) and Hs683 (C) cells. Immunofluorescence (IF) analysis of galectin-1 expression levels in U87 day 7 post-transfection (B) and Hs683 day 5 post-transfection (D) using scrambled or anti-galectin-1 siRNA. Top rows are the corresponding bright fields to the IF images.

Galectin-1-targeted siRNA induces both a decrease of integrin-β1 at the edges of the cell membrane and an increase in the intracellular accumulation of integrin-β1. A. U87 control wild-type cells (Aa,Ab) and U87 cells containing stably transfected antisense galectin-1 vectors (Ac) were co-stained under non-permeabilized conditions for integrin-β1 [the red staining at the tip of actin stress fibers (in green fluorescence)], fibrillar actin (green) and globular actin (red staining inside the cells). Ab. Higher magnification of the control wild-type (Aa) image. B. U87 control wild-type cells (Ba) and U87 cells containing stably transfected antisense galectin-1 vectors (Bb) were stained under permeabilized conditions for integrin-β1 only (in green), without any staining to reveal actin (Ba,Bb). C. U87 and Hs683 cells were transiently transfected with either scrambled siRNA (scr) or siRNA targeted against galectin-1 (siGal1). Cells were stained for integrin-β1 (in red) under both permeablized and non-permeabilized conditions on either day 5 post-transfection (Hs683) or day 7 post-transfection (U87).

Western blot (WB) and immunofluorescence (IF) analysis of vimentin and protein kinase C epsilon (PKCε) protein expression levels; galectin-1-targeted siRNA increases only vimentin protein levels in Hs683, induces increased perinulear amassment of both PKCε and vimentin, and diminishes the diffuse cytoplasmic staining of PKCε. WB analysis of PKCε and vimentin protein expression levels in U87 (A) or Hs683 (B) cells untreated (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) at days 5, 7 and 9 post-transfection. IF analysis of PKCε localization in U87 (C) or Hs683 (D) scr or siGal1 at either day 5 (Hs683) or day 7 (U87) post-transfection. IF analysis of vimentin localization in U87 (E) or Hs683 (F) cells scr or galectin-1 siRNA-transfected (siGal1) at either day 5 (Hs683) or day 7 (U87) post-transfection. In all IF figures right columns represent the corresponding bright field images.