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. 2019 Sep 18;5(9):eaax6352. doi: 10.1126/sciadv.aax6352

Fig. 3. SLC36A1 overrides CDK4/6i-induced senescence.

Fig. 3

(A) Clustering of SLC transporters from 1205Lu cells treated with or without palbociclib (1 μM) for 1 day and 1205CR1, 1205CR2, 1205CR6, and 1205CR7 cells proliferating with palbociclib (1 μM) for 8 days. (B) qPCR analysis of samples from 1205Lu cells treated with or without palbociclib (1 μM) for 8 days or in 1205CR1, 1205CR2, 1205CR6, and 1205CR7 cells proliferating with palbociclib (1 μM) using a set of primers for SLC36A1. Data were normalized by GAPDH and represent means ± SD. *P < 0.01 (two-tailed Student’s t test; n = 3). (C) Western blot analysis of lysates from (B) for SLC36A1 and β-actin. The numbers indicate quantifications of SLC36A1 determined by SLC36A1/β-actin ratio. (D) Representative images of IHC staining of sections from xenograft tumors treated with either vehicle or palbociclib (1 μM) or tumors resistance to CDK4/6i for SLC36A1. Scale bars, 100 μm. The numbers indicate quantification of SLC36A1 intensity determined by IHC scoring (see Materials and Methods) from three independent experiments. (E) Western blot analysis of lysates from 1205Lu cells introduced with shcontrol or shSLC36A1 using antibodies to SLC36A1 and β-actin. (F) Clonogenic colony formation assay of cells from (E) treated with or without palbociclib (1 μM) for 8 days. The numbers indicate quantification of colonies from three independent experiments. (G) Western blot analysis of lysates from 1205Lu cells infected with empty vector (control) or SLC36A1 using antibodies to SLC36A1, p-S6K, and β-actin. (H) Clonogenic colony formation assay of cells from (G) treated with rapamycin (50 nM), palbociclib (1 μM), or rapamycin (50 nM) + palbociclib (1 μM) for 8 days. The numbers indicate quantification of colonies from three independent experiments. (I) Representative images of SA-β-gal staining from (G) treated with palbociclib (1 μM) for 8 days. (J) Quantification of SA-β-gal–positive cells from (I). Data represent means ± SD. *P < 0.01 (two-tailed Student’s t test; n = 3). (K) Western blot analysis of lysates from WM983B, WM239A, 451Lu, and WM3918 cells infected with empty vector (control) or SLC36A1 using antibodies to SLC36A1 and β-actin. (L) Quantification of SA-β-gal–positive cells from (K) and parental cells treated with or without palbociclib (1 μM) for 8 days. Data represent means ± SD. *P < 0.01 (two-tailed Student’s t test; n = 3). (M) Western blot analysis of lysates from 1205Lu, 1205CR1, 1205CR2, 1205CR6, and 1205CR7 cells transfected with siControl or siSLC36A1 using antibodies against SLC36A1, p-S6, and β-actin. (N) Quantification of SA-β-gal–positive cells after 8 days after transfection; data represent means ± SD, *P < 0.01 (two-tailed Student’s t test; n = 3).