siRNAs designed to target mouse-specific lncRNAs lead to cell death in human CRC cells (A, B) Representative microscopic graphs of HCT116, DLD1, and CT26 cells taken 48 hours after transfection with siNC, siRNAs targeting mouse-specific lncRNAs Gm38195 or Gm48223, respectively. Apoptotic and pyroptotic cells were denoted by red or yellow arrowheads, respectively. “siNC” was a 19bp siRNA designed by RiboBio with no targeting sequence, acting as a negative control. “siRNA-1/2/3” meant three 19bp siRNAs targeting different sequences of the lncRNA as indicated. (C, D) Percentages of the area covered by living cells under indicated treatments, three independent perspectives were randomly selected for measurement by using Image J. Scale bar, 100μm. (E -H) Total cell death was determined by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant difference.

Targeting human-specific lncRNAs with siRNAs fails to trigger cell death in mouse cancer cells (A, B) Images representing microscopic views of CT26, B16F10, and DLD1 cells treated with specific siRNAs. (C, D) Percentages of the area covered by living cells. Scale bar, 100μm. (E -H) Total cell death was assessed by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant difference.

siRNAs targeting rat-specific lncRNAs exhibit specific cytotoxicity against human cancer cells without affecting mouse cancer cells (A, B) Representative microscopic pictures of HCT116, DLD1, and CT26 cells under indicated treatments. Apoptotic and pyroptotic cells were denoted by red or yellow arrowheads, respectively. (C, D) Percentages of the area covered by living cells. Data were presented in the format of mean ± SD. Scale bar, 100μm. (E -H) Percentages of total cell death determined by flow cytometric analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant difference.

siRNAs targeting zebrafish-specific lncRNAs exhibit specific cytotoxicity against human cancer cells without affecting mouse cancer cells (A, B) Representative microscopic pictures of HCT116, DLD1, and CT26 cells treated as indicated. Apoptotic and pyroptotic cells were denoted by red or yellow arrowheads, respectively. (C, D) Percentages of the area covered by living cells under specified treatments. Data were presented in the format of mean ± SD. Scale bar, 100μm. (E -H) Percentages of total cell death determined by flow cytometric analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant difference.

siRNAs targeting zebrafish-specific lncRNA BX323557.1 do not cause death in normal human or mouse cancer cells (A) Schematic of genomic alignments of lncRNA BX323557.1 with human and mouse genome through UCSC Genome Browser. (B) Representative microscopic pictures of FHC and NCM460 cells treated with the specified siRNAs and (C) Percentages of the area covered by living cells. Scale bar, 100μm. (D -K) Total cell death was detected by flow cytometry in HCT116, DLD1, NCM460, and CT26 cells treated as indicated. All the data are presented in the format of mean ± SD from three independent experiments. ****p < 0.0001, ns, no significant difference.

siRNAs designed to target zebrafish-specific lncRNA BX323557.1 prompt apoptosis or pyroptosis in human CRC cells (A) Representative microscopic pictures of HCT116 and DLD1 cells treated with siBX323557.1. Apoptotic and pyroptotic cells were denoted by red or yellow arrowheads, respectively. Scale bar, 100μm. (B) Comparison of LDH levels in the cultural supernatant between two human CRC cell lines. (C) Morphologies observed by the transmission electron microscope of HCT116 cells under indicated treatments. Scale bar, 2μm. (D, E) The expression of protein PARP, GSDME, caspase-3, and the corresponding cleaved segments in HCT116, DLD1, and NCM460 cells treated with specified siRNAs. (F) Quantified statistics of immunoblotting analyzed by Image J. The level of α-tubulin is applied as an internal control. All the data are presented in the format of mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant difference.

siRNAs targeting zebrafish-specific lncRNA BX323557.1 elicit cell death by triggering an IRF3-independent immune response. (A) Off-target genes of siBX323557.1-1/2/3 in the human genome predicted by database pita, miranda, targetScan, and RNAhybrid. (B) “1 VS 2 VS 3” referred to the prediction of shared off-target genes of siBX323557.1-1/2/3 in the human genome. (C) Expression of dsRNA sensors detected by immunoblotting. (D) The relative mRNA levels of type-I interferon genes. (E) Representative bright-field pictures of NCM460, CT26, and HCT116 cells under specified treatments. Scale bar, 100μm. (F) Total cell death was determined by the flow cytometric analysis. (G) The protein expression of IRF3 and p-IRF3 in HCT116 cells upon the indicated treatments. p-IRF3: phosphorylated IRF3. (H) The verification of silencing efficiency of siIRF3 by immunoblotting and qRT-PCR. (I) The comparison of normalized cell death caused by siBX323557.1-2 with or without IRF3 silencing. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, no significant difference.