Fig. 3

LINC00115 links SETDB1 and PLK3 to regulate HIF1α expression. A Representative image of silver-stained PAGE gels showing separated proteins that were pulled down using biotin-labeled LINC00115. LINC00115 and anti- LINC00115 RNA were biotinylated by in vitro transcription, refolded, and incubated with MDA-MB-231 BCSCs total cell lysates. B WB of the proteins from antisense LINC00115 and LINC00115 pull-down assays. C Effects of LINC00115 KD on SETDB1 associated with PLK3 and HIF1α expression in MDA-MB-231 and B549 BCSCs. D Effects of LINC00115 KD on HIF1α expression with or without ectopic expression of HA-SETDB1 or/and Flag-PLK3 in MDA-MB-231 and B549 BCSCs. E A schematic model of LINC00115 functions in metastasis. LINC00115 may function as a scaffold lncRNA to recruit SETDB1 and PLK3 to activate the HIF1α signaling pathway. F Schematics of PLK3 full length (WT, 1-646aa), D1 (N-terminal, 1-315aa, contain Kinase domain (KD) mutant, and D2 [C-terminal, 316-646aa, contain polo box domain (PBD)] mutant plasmids. G Immunoblot analysis (IB) detection of SETDB1 interaction with PLK3 was immunoprecipitated with anti-Flag magnetic beads in HEK-293T cells transfected with the HA-SETDB1 and Flag-PLK3 constructs. H Schematics of SETDB1 full length (WT, 1-1291aa), M1 (N-terminal, 1-667aa, contain Tudor domain) mutant, and M2 (C-terminal, 672-1291aa, contain SET domain) mutant plasmids. I. IB of SETDB1 interaction with PLK3 and PLK3 methylation was immunoprecipitated with anti-HA magnetic beads in HEK-293T cells transfected with the Flag-PLK3 and HA-SETDB1 constructs