Fig. 4

Examination of the effects of miR-193b overexpression on active caspase 3, cell morphology and Cyto-ID. a Active caspase 3 levels were assessed by FACS analysis. (i) KYSE450 cells were transfected with negative control mimic or miR-193b mimic (5 nM) and the following day were treated with 5-FU (10 μM or 30 μM) for 48 h. An upward shift in the levels of Alexa-Fluor 488 represented an increase in the number of cells with activated caspase 3 (percentage of cells with activated caspase 3 is shown). Upper panels – negative control transfected cells treated with 10 μM (middle) or 30 μM 5-FU (right panel). Lower panels – miR-193b transfected cells treated with 10 μM (middle) or 30 μM 5-FU (right panel). (ii) OE21 cells were treated with 30 μM 5-FU for 48 h (lower panel). b Morphological features of negative control mimic or miR-193b (5 nM) transfected KYSE450 cells. Twenty four hours following transfection, cells were treated with 5-FU (10 μM) for 24 h and compared to untreated transfected controls. Autophagic cells are indicated with arrows (×40 magnification). c KYSE450 cells were transfected with negative control mimic or miR-193b mimic (5 nM). Twenty-four hours following transfection, cells were treated with 5-FU (10 μM) for 24 h and then assessed for autophagy induction using the Cyto-ID autophagy detection assay. Representative image of FACS analysis of Cyto-ID levels of (i) untreated, negative control mimic and miR-193b transfected cells and (ii) untreated, negative control mimic and miR-193b mimic cells treated with 10 μM 5-FU. Right hand panel - mean fluorescence intensity of all treatments. Asterisks indicate statistical significance determined by t-test (*p < 0.05, **p < 0.01)