Figure EV5. Incucyte analysis with representative images and ex vivo analysis.

(A) Anergic and responsive cells were treated with either Egr2 or NS siRNA, and Incucyte-based tumor lysis assay was performed on 721.221 HLA-Cw7 target cells expressing mCherry. P value was cumulatively calculated for every time point using one-way ANOVA, and Tukeys’ post hoc test was used for multiple comparisons, as indicated in the graph (*P < 0.05). The analysis was performed for nine individual fields per image (16 images) (with ~N = 50 cells in each field). Green line: anergic NS siRNA; red line: anergic Egr2 siRNA; blue line: responsive NS siRNA. Anergic and responsive cells were transfected with EGR2 siRNA or NS siRNA, seeded with 721.221 HLA-Cw7 expressing mCherry cells at an E:T ratio of 10:1, and subjected to Incucyte imaging and analysis; the decrease in fluorescence intensity was measured, and images from the indicated time points are shown. The values were normalized to 721.221 Cherry only—no effector control. Right panel: Images showing cell fluorescence at different time points (scale bar: 200 µm). (B) Human CML OTS prepared using K562 CFP cells in Matrigel (1:1–v/v%), were seeded with anergic or responsive pNK cells at an E:T ratio of 5:1. After 6 h, NPs encapsulating Egr2 siRNA or NS siRNA were added to the OTS, and the decrease in fluorescence intensity was monitored and measured; the images along with the fluorescence intensity at the respective time points are shown (numbers on top right of each frame). The decrease in fluorescence over time shows the enhanced cytotoxic activity of anergic cells following Egr2 siRNA treatment (left panel: middle) similar to the responsive cells treated with NS siRNA (left panel: bottom). Whole well images of the OTS at 48 h are presented. Right panel: Images showing cell fluorescence at different time points (Scale bar: 200 µm). (C) The tumors were excised on day 27, a single-cell suspension was made, and cells were stained for intracellular Egr2. The pNK cells were distinguished based on FSC vs SSC, and PE content, reflecting nanoparticle incorporation. Representative histogram showing Egr2 expression levels. Blue line shows the EGR2 levels in the group treated with control NP (NS siRNA), and the red line shows the group receiving Egr2 siRNA-NP. (D) The dissociated tumors were then subjected to Annexin V staining for apoptotic cells. Representative histogram on the left panel shows the apoptosis of the tumors from each group (one mouse representative of three independent repeats). The right panel shows a graph summarizing the tumors obtained from three different mice. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA with Tukeys post hoc test after normalization of the values to the “tumor only” group, which served as the control. Source data are available online for this figure.