Fig 2: Analysis of Hspb1 expression in TSC complex subunit 2-deficient kidney tumor cells (MKOC). (A) MKOC cells were treated with 20 nM Rapa or vehicle only (DMSO) for 48 h. Cell proliferation was then analyzed by XTT assays. Data are representative of three independent experiments. Each experiment was performed with three independent culture wells. The data are presented as the mean + SD. ***P<0.001 compared with Rapa. (B) MKOC cells were treated with 20 nM Rapa or vehicle only (DMSO) for 24 h. Total cell lysates were analyzed by western blotting with the indicated antibodies (Hspb1, pHspb1, pS6, S6 and ßactin). (C) MKOC cells were treated with 20 nM Rapa or vehicle only (DMSO) for 24 h. Total cell lysates were analyzed by western blotting with the indicated antibodies (Hspb1, Hsp90, Hsp70 and a-tubulin). Hsp90, heat shock protein 90; Hsp70, heat shock protein 70; Hspb1, heat shock protein family B (small) member 1; MKOC, MKOC1-277; p, phosphorylated; Rapa, rapamycin.

Fig 3: Analysis of MKOC1-277 cells stably expressing Hspb1 and its mutants. (A) Total cell lysates were analyzed by western blotting with the indicated antibodies. Cells were treated with (B) vehicle only or (C) 20 nM rapamycin for 48 h. Cell proliferation was analyzed by XTT assays. Data are representative of three independent experiments. Each experiment was performed with three independent culture wells. The data are presented as the mean + SD. *P<0.05 compared with empty vector; #P<0.05 compared with wild-type Hspb1. Empty, control empty vector; A-mutant, phosphorylation-resistant S86A-mutant; D-mutant, phosphorylation-mimetic S86D-mutant; Hspb1, heat shock protein family B (small) member 1; p, phosphorylated; W-Hspb1, wild-type Hspb1 overexpression.

Fig 4: Analysis of MKOC cells transfected with Hspb1 siRNA. (A) MKOC cells were treated with Hspb1 siRNA for 48 h. Total cell lysates were analyzed by western blotting with the indicated antibodies. (B) MKOC cells were treated with siRNA (+) or siRNA (-) for 24 h. Subsequently, each group of cells was treated with 20 nM Rapa or vehicle only for 48 h. The start point of the drug treatment was defined as Day 0. Cell proliferation was analyzed by XTT assays. Data are representative of three independent experiments. Each experiment was performed with three independent culture wells. The data are presented as the mean + SD. *P<0.05 compared with siRNA (-) and Rapa (-); #P<0.05 compared with siRNA (+) and Rapa (-). Cont, control; Hspb1, heat shock protein family B (small) member 1; MKOC, MKOC1-277; siRNA, small interfering RNA; siRNA (+), Hspb1 siRNA; siRNA (-), control RNA; Rapa, rapamycin; Rapa (-), vehicle only.

Fig 5: Confirmation of Hspb1 expression in HeLa and COS7 cells. Cells were treated with 20 nM Rapa (+) or vehicle only (-) for 24 h. Total cell lysates were analyzed by western blotting with the indicated antibodies. Hspb1, heat shock protein family B (small) member 1; p, phosphorylated; Rapa, rapamycin.