Fig 2: Expression and roles of p16INK4a, p21Cip1 and p27Kip1 in the response of lung adenocarcinoma cell line models to long-term treatment with Dex. H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days and whole cell lysates were probed by western blot for p16INK4a, p21Cip1 and p27Kip1 with GAPDH as the loading control (Panel A). H1299GR Clone 4 cells were transduced with either non-targeted control shRNA or P21Cip1 shRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were extracted and probed by western blot for p21Cip1, p27Kip1 and GAPDH (loading control) (Panel B). In parallel, cells treated as described for Panel B, were plated for colony formation in 6-well plates in triplicates without further treatment (Panel C). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27Kip1 siRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were probed by western blot for p27Kip1 or GAPDH (loading control) (Panel D). In parallel, cells transfected as described for Panel D, and treated with Dex (100 nM) were plated on days 0 and 5 to measure colony formation in 6-well plates in triplicates without further treatment (Panel E) and also harvested on days 0 and 3 of treatment for measuring cell size (Panel F). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27Kip1 siRNA and 24 h later they were treated with Dex (100 nM) or vehicle for 3 days and the cells were stained to assess expression of β- galactosidase (blue staining) (Panel G, left); in parallel, the treated cells were lysed and analyzed by western blot for p27 expression (Panel G, right). H1299GR Clone 4 cells were transduced with either non-targeted control vector or pLenti-p27 expression vector and after the indicated number of days, whole cell lysates were probed by western blot for p27Kip1 or GAPDH (loading control) (Panel H). In parallel, cells were harvested for measuring the cell size (Panel I) and also plated to measure colony formation in 6-well plates in triplicate (Panel J). Panel C: *P, 0.0004; **P, 0.0006; §P, 0.0008; ‡P, 0.00015. Panel E: *P, 0.016. Panel J: *P, 0.015; §P, 0.00006; ‡P, 0.00007.

Fig 3: FAM46C inhibited cell growth of DU145 cells via the PTEN/AKT signaling pathway. After DU145 cells were transduced with pLVX-Puro-FAM46C with or without SF1670 or IGF-1, the cell proliferation (A, B), cell cycle (C), cell apoptosis (D) and expression of PTEN, P27, cleaved Caspase-3, AKT and p-AKT (E, F) were measured by CCK-8, colony formation, flow cytometry and western blot, respectively. **P<0.01, ***P<0.001 compares with vector; ###P<0.001 compared with FAM46C.

Fig 4: LIMK2 overexpression in UM‐UC‐3 cells promotes proliferation and cell cycle progression, inhibits apoptosis, and affects expression of multiple cell cycle regulators. Cells were stably transfected with LIMK2‐targeted shRNA (sh‐LIMK2), scrambled control siRNA (Scramble), LIMK2 overexpression vector (LIMK2), or empty vector (Vector). (a and b) Cell proliferation measured by MTT and EdU assays. (c) Cell cycle distribution of UM‐UC‐3 cells measured by flow cytometry. (d) Cell apoptosis measured by TUNEL assays. (e) Western blotting of LIMK2, p27, cyclinD1 and ki67. β‐actin was used as a loading control.

Fig 5: Expression of IGF1-R, p27, c-MYC, p53 and p-AKT in cells transfected with either a non-specific mimic (pm-neg) or miR-7 mimic (pm-7).GAPDH was used as a loading control.