Overexpressing CUL3 in HNSC cell lines suppresses tumor growth. (A) Somatic missense mutations in CUL3 are shown as spheres in the structure of a homologous protein (chain C of PDB: 2HYE, sequence identity = 39.82%), and the EA score of the given mutation is shown in parentheses. The importance of position was evaluated by ET and is represented by a color scale from red (more important) to green (less important). (B) Clonogenic assay following infection of the cell lines UMSCC25, HN31, UMSCC22A, and MDA1386LN with lentivirus to express wild-type CUL3 or the control gene LacZ. (C) Western blot shows the ectopic expression of wild-type CUL3 after lentivirus infection. (D) Cell cycle distribution for the UMSCC25 cell line following expression of CUL3. (E) PARP levels, a marker of apoptotic death, increase in UMSCC25 cells expressing CUL3; (F) levels of NFE2L2 (NRF2), the HNSC-driving protein, are reduced. Overexpression of DUSP16 inhibited cell proliferation, colony formation, migration, and induced apoptosis by inhibition of JNK pathway. (G) DUSP16 overexpression was assessed by induction with doxycycline (1 µg/mL) to stable HEK293 and BT474 cells containing inducible DUSP16 construct. (H) Overexpression of DUSP16 inhibited cell proliferation of established cells. (I) Clonogenic assay: Overexpression of DUSP16 inhibited colony formation of HEK293 and BT474 cells. The experiment was performed in triplicates, and the density of the stained cells was measured at 630 nm after extraction with 10% acetic acid. (J) In vitro scratch assay. Cells were plated and incubated until confluent and then scratched with pipette tip and further incubated to compare recovery of scratched area. (K) Overexpression of DUSP16 induced apoptosis in stable BT474 cells. (L) Overexpression of DUSP16 inhibited JUN phosphorylation in stable HEK293 and BT474 cells. Overexpression of DUSP16 induced down-regulation of JUN and FOS transcription in HEK293 cells (M) and BT474 cells (N). (O) Depletion of DUSP16 by CRISPR antagonized cell growth inhibition by overexpression of DUSP16. (*) P-value < 0.05, (**) P-value < 0.005, and (***) P-value < 0.001.
