Analysis of the RNA sequencing data provided further evidence of RB1/E2F activation, as E2F target genes were more highly expressed in the absence of FLCN. Furthermore, upon FLCN knockdown, we observed a modest reduction in the G1 population of cells, suggesting a potential increase in proliferative drive through G1/S in the absence of FLCN. The high protein levels of CCND1 and RB1 hyperphosphorylation in long-term FLCN-knockdown HK2 tumor spheroids likely contributed to their observed tumorigenicity. This evidence concerns the gene RB1 and neoplasm.