Enrichment analysis and validation of the unfolded protein response (UPR) system’s modulation. (A) Image adapted from Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (full image is provided in supplemental Figure 18) of differentially expressed genes in RP11-350G8.5 KO ABZB cells revealing a network of genes involved in the UPR in endoplasmic reticulum (ER). (B-F) Western blot (WB) analysis conducted on proteins extracted from ABZB and AMO-1 GFP⁺ sorted cells after RP11-350G8.5 KO, or overexpression (UP) or transduced with the SCRAMBLE vector, or following bortezomib treatment. (G) Distribution of SCRAMBLE and KO ABZB and AMO-1 cells stained with annexin V/7AAD to assess the percentage of apoptosis following the KO. The fold increase of annexin V⁺/7AAD⁺ late apoptotic events was 2.7 for ABZB KO cells and 1.8 for AMO-1 KO cells, compared with SCRAMBLE-transduced cells. (H, left) Representative images from a FISH analysis conducted with the probe targeting the lncRNA RP11-350G8.5 conjugated with TAMRA dye (red signal) and the antibody against CRT protein, whose secondary antibody is conjugated with FITCH (green signal). Images were acquired with a DMI6000-AF6000 Leica microscope (magnification ×63). (H, right) Membrane expression of CRT on ABZB cells transduced with SCRAMBLE (gray curve) or KO vector (green curve), detected through flow cytometry (7AAD-negative cells were gated to exclude dying cells from this analysis). The fold change of the median fluorescent intensity (MFI) was calculated to quantify the increase of CRT exposure to the cell membrane following KO, with respect to scramble cells. (I) Representative images of immunofluorescent assay show ABZB tumor cells in green and monocyte-derived dendritic cells (MoDCs) in red. After RP11-350G8.5 KO, there is an evident increase of cancer cells engulfed in MoDCs, as indicated by arrows. (Enlarged images of phagocytosed cells are reported near the pictures with magnification ×20.) The images were obtained with a DMI6000-AF6000 Leica microscope. A quantification of engulfed cells following KO was provided by a flow cytometric analysis (supplemental Figure 19). (J) Hematoxylin and eosin (H&E) and p-PERK immunohistochemistry staining of retrieved tumors from animals engrafted with ABZB SCRAMBLE or ABZB RP11-350G8.5 KO cells revealed the high grade of engrafted tumors, based on histologic evaluation of nuclear atypia, necrosis, and mitotic pattern. Visualization was performed with a Leica DM 2500 optical microscope (magnification ×20) (upper panel). WB analysis of p-PERK and the downstream effector p-eIF2 α on whole protein extracted from tumors retrieved from mice 16 days after engraftment (lower panel).