Supplementary Methods from miRNA-130a Targets &lt;i&gt;ATG2B&lt;/i&gt; and &lt;i&gt;DICER1&lt;/i&gt; to Inhibit Autophagy and Trigger Killing of Chronic Lymphocytic Leukemia Cells

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section_content: (TO-PRO-3) and 520 nm (GFP) for each sample. The acquired images were analyzed with IDEAS software (Amnis). First, a scatter plot of aspect ratio/area was used to gate for single cells. Second, a gradient RMS (root mean square for image sharpness) histogram was used to gate for cells in focus. Third, the intensity of TO-PRO-3 staining was used to exclude dead cells. On the remaining living, single cells in focus, image analysis was performed to determine the formation of autophagosomes by the extent of GFP-LC3 clustering. To do that, a spot mask was generated to automatically recognize the cellular regions containing GFP clusters. The level of fluorescence clustering was extracted from the individual cellular images and was represented by the bright detail intensity R3 feature. This feature computes the intensity of localized bright spots within the masked area in the image. The bright detail intensity was quantified at the cell population level for each sample and the data was analyzed using MATLAB (Mathworks, Inc., Massachusetts, USA). First, a lillietest for normality distribution was performed on the cellular bright detail intensities, showing that the data are non-normally distributed. Therefore, the data were analyzed using a non-parametric description derived from the Kolmogorov-Smirnov (K-S) statistics. The bright detail intensity distributions were shown as cumulative distribution functions. We defined that the autophagic flux can be described at the population level by the difference between the cumulative distribution functions between bafilomycin-treated cells and untreated cells. The difference between 2 cumulative distributions was calculated according to K-S statistics, by subtracting the values for each feature channel (histogram bin value). The resulting autophagic flux distributions were normalized to the maximum channel of the sample with the highest autophagic flux (set as 100%). Normalized autophagic flux distributions of 4 independent experiments were averaged and the data shown as mean ± SEM for each channel. In addition, a Student's t-test was performed for each channel in order to determine the statistical significance of the population frequency changes in function of the autophagic flux.