Fig. 6

Analysis of morphology, autophagy levels and HMGB1 release from recovering 5-FU treated miR-193b overexpressing cells. KYSE450 cells were transfected with negative control mimic or miR-193b (5 nM). Twenty four hours following transfection, cells were treated with 5-FU (10 μM) for 24 h, following which these cells were allowed to recover in drug free media for 96 h. a Morphological features of negative control mimic or miR-193b transfected KYSE450 cells which had been treated with 5-FU for 24 h and left to recover 96 h. Autophagy and non-apoptotic/type II cell death are shown by arrows and arrowheads respectively (×40 magnification). b Cyto-ID analysis of the KYSE450 cells at the 96 h recovery time-point. Upper panel shows FACS analysis of untreated, miR-193b mimic transfected cells in the presence or absence of 5-FU (10 (i) or 30 μM (ii)) and negative control mimic 5-FU treated cells (10 μM (i) or 30 (ii)). Negative control mimic (fluorescence levels equivalent to untreated) and 5-FU alone treated cells (fluorescence levels equivalent to negative control mimic treated with 5-FU) were omitted from these images to aid visualisation of the other treatments. The lower panels show corresponding mean fluorescence intensity of all treatments. (C) At the 96 h recovery time-point, media was removed from all treated cells to assess HMGB1 levels. HMGB1 levels were quantified using a HMGB1 ELISA. Asterisks indicate statistical significance determined by t-test (*p < 0.05, **p < 0.01, ***p < 0.001)