Skip to main content
. 2021 Jun 18;11:12893. doi: 10.1038/s41598-021-92223-x

Figure 5.

Figure 5

Effect of the Downregulation of HML-2 on AT/RT. (a–d) A decrease in HML-2 expression leads to reduced cellular aggregates in CHLA 02 AT/RT cells. (a) HML-2 env transcript levels in CHLA 02 cells 48 h post transfection with either shRNA targeting env or CRISPRi targeting HML-2 LTR. (b) CHLA 02 cells transfected with CRISPRi + gRNA have reduced aggregate size following reduced expression of HML-2 mRNA at 24 h, 48 h, and 72 h post transfection. (c) CHLA 02 cells at 72 h post transfection with either pcDNA or (d) with CRISPRi + gRNA. (e–i) Downregulation of HML-2 results in AT/RT cell death and reduced cell aggregates. (e) CHLA 02 and CHLA 04 cells transfected with CRISPRi with or without gRNA targeting HML-2 LTR results in different proportions of viable cells at 96 h post transfection. (f) Percent of control (siRNA non-targeting) env transcription following transfection with siRNA targeting HML-2 env in CHLA 02 at 24 h. (g) CHLA 02 cell aggregates were reduced in size after 72 h following siRNA transfection targeting HML-2 env. (h) CHLA 02 at 72 h post transfection with both non-targeting siRNA and (i) cells treated with HML-2 env siRNA. (j–m) Lentiviral vectors with CRISPRi + gRNA targeting HML-2 and shRNA targeting HML-2 env result in decreased AT/RT viability and a reduction in HML-2 env protein expression. (j,k) CHLA 02 cytotoxicity at multiple time points post-transduction with lentivirus expressing either a CRISPRi construct with CRISPRi + gRNA targeting HERV-K (HML-2) LTR5_Hs or with CRISPRi without gRNA. (l) Percent viable cells 48 h post-transfection shRNA targeting HML-2 env. (m) env protein post-transfection as a percent of control transfection (either with CRISPRi and no gRNA or shRNA (−)). Graphs generated with Prism v9. [Error bars represent SEM. Statistics in Supplemental Table S6].