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Fig. 5. SNARE downregulation – differential effects on enlargeosome exocytosis. (A) Western blots of VAMP4, Stx6 and VAMP8 in PC12-27 (left) and SH-SY5Y (right) cells, transfected (+) or not (–), with siRNAs. Notice the decrease (by 
50-60%) in the levels of the respective SNARE proteins in the PC12-27 populations transfected with siRNAs for VAMP4, Stx6 and VAMP8; in SH-SY5Y cells, which were all transfected with VAMP4 siRNA, notice the decrease of VAMP4 (by 40%), with no change in VAMP2 levels. The top band (calnexin) is shown to certify that equal protein loads were seeded in the slots. (B,D,F,H) The correlation between surface desmoyokin (d/A), documenting enlargeosome exocytosis, and the total level of individual SNAREs in single PC12-27 (B,D,F) and SH-SY5Y (H) cells non-transfected (blue signs) or transfected (red signs) with the siRNAs specific for the indicated SNAREs and then stimulated with ionomycin (3 µM, 1 minute) is shown. After fixation, the cells were first immunolabelled for surface desmoyokin, then permeabilized and immunolabelled for VAMP4 (circles, B and H), Stx6 (triangles, D) and VAMP8 (squares, F). (B) Notice that, in the population of PC12-27 cells exposed to VAMP4 siRNA (red circles), cells with levels of the SNARE that were distinctly lower than those of the untreated cells (blue circles) (by 90.3±1.7% on average in a group of 20 cells) exhibited a parallel decrease of enlargeosome exocytoses, revealed by the low level of surface appearance of desmoyokin (decreased by 89.9±1.8% compared with non-transfected cells). (H) A similar, although less intense, result was obtained in the SH-SY5Y cells transfected with the human siRNA (–75.2±5.8% for SNARE level and –64.8±7.5% for desmoyokin). (D,F) In PC12-27 cells downregulated for Stx6 (red triangles), the results were closer to those obtained in the same cells with the siRNA for VAMP4 (–79.9±5.6 for SNARE level and –84.6±5.6% for desmoyokin, D), whereas in those downregulated for VAMP8 (red squares, F), the drop in the level of SNARE (–94.7±1.9%) was dissociated from the surface appearance of desmoyokin, which was almost unchanged (–7.8±7.6%). (C,E,G,I) Examples of PC12-27 (C,E,G) and SH-SY5Y (I) cells in the populations of B, D, F and H, i.e. exposed to the siRNAs of VAMP4, Stx6 and VAMP8 and then stimulated with ionomycin, chosen because of their high (C,E,G,I) and low (C',E',G',I') labelling for the three SNAREs. Notice the clear desmoyokin surface labelling of C, E, G and I (yellowish in C and I because of the co-discharge of VAMP4) and its lack in C', E' and I' but not in G'. Scale bar: 5 µm.
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