Fig. 2: The long non-coding RNA HHIP-AS1 is functionally required in human SHH-driven brain tumors.

a The relative gene expression levels of HHIP-AS1 and the SHH target gene GLI1 were tested in tumor cell lines (Daoy and CHLA-266) and in primary tumor cell cultures (HHU-ATRT1) upon pharmacological activation (SAG, Smoothened agonist) or inhibition (CYC, cyclopamine) of the SHH pathway. b Relative gene expression levels of indicated genes as measured by qRT-PCR upon transient knockdown of GLI1 and GLI2 in the depicted cell models normalized to control (si-negative-POOL; gene expression of target genes were normalized to housekeeping genes: HPRT, GUSB and PPIA). c Proliferation rate of Daoy, CHLA-266 and HHU-ATRT1 was measured by EdU incorporation upon transient (si-HHIP-AS1) or stable HHIP-AS1 knockdown normalized to control. d Self-renewal capacity of Daoy, CHLA-266 and HHU-ATRT1 was measured by colony formation assay upon transient (si-HHIP-AS1) or stable (sh-HHIP-AS1#1 and sh-HHIP-AS1#2) HHIP-AS1 knockdown normalized to control. In panel c + d corresponding controls (either with si-negative-POOL or sh-scr transfected Daoy, CHLA-266 and HHU-ATRT1 cells) were set to 100% and levels of knockdowns were calculated accordingly. e Proliferation rate of primary SHH MB cultures derived from freshly resected tumors (n = 2 patients) measured by EdU incorporation upon transient knockdown of HHIP-AS1 (si-HHIP-AS1) normalized to control (si-negative-POOL). f Cell viability of these primary SHH MB cultures derived from freshly resected tumors (n = 2 patients) measured by CellTiter-Glo upon transient knockdown of HHIP-AS1 (si-HHIP-AS1) normalized to control (si-negative-POOL). g Proliferation rate of SHH MB PDX cells (ICN-MB12) determined by BrdU incorporation and Ki67 immunostaining after transient knockdown of HHIP-AS1 (sh-HHIP-AS1#1) normalized to control. Bar graphs of panels a + b are presented as the mean ± SD, panels c–g are presented as the mean ± SEM of at least three independent experiments and corresponding controls were set to 100%. Student’s two-sided t-test; ***p < 0.001; **p < 0.01; *p < 0.05. Source data and exact p-values are provided as a “Source Data file”.