A Tsc2-deficient kidney tumor cell line, MKOC1-277 (MKOC) was established in our laboratory from the Tsc2 knockout mouse and, together with its derivatives, had been used in various researches (30-35). MKOC cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin. Rapamycin (Sigma-Aldrich, Darmstadt, Germany) treatment was performed with dimethylsulfoxide (DMSO) as a vehicle. HeLa, COS7 and Plat-E cells (COSMO BIO, Tokyo, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 1 µg/ml of puromycin, 100 U/ml of penicillin and 100 µg/ml of streptomycin. In the case of Plat-E cells, 10 µg/ml of blasticidin was also added. All cell cultures were performed at 37˚C with 5% CO₂. SB203580 was purchased from Sigma.

Cell proliferation was assessed with the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay system (Roche, Basel, Switzerland) in accordance with the manufacturer's guidelines using 96-well plates. To 200 µl of culture medium, XTT reagent (50 µl) was added to each well and incubated for 4 h. Measurement of the absorbance at 450 nm (reference wavelength of 650 nm) was performed with a microplate reader (Benchmark Plus-microplate Spectrophotometer; Bio-Rad, Hercules, United States).

Cell lysates were prepared in Laemmli's sample buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determination by DC Protein Assays (Bio-Rad). Equal amounts of proteins were separated by SDS-PAGE using standard methodology and proteins were transferred to nylon membranes (Millipore, Burlington, United States). For blocking and antibody reactions, we used 1% skimmed milk/Tris buffered saline supplemented with 0.05% of Tween 20 (TBST). Anti-mouse or anti-rabbit Envision HRP-conjugate (DAKO, K4001 or K4003, respectively) was used as secondary antibodies. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, United States) or Immobilon Forte (Millipore) was used for the development of signals. A Bio-Rad ChemiDoc MP imaging system was used for the detection and analysis of signals. We used several different primary antibodies: anti-HSP27 (#sc13132), -pHSP27(S82) (#sc166693), -Hsp70 (#sc24, #sc59569), and Hsp90 (#sc13119); these antibodies were purchased from Santa Cruz Biotechnology (Dallas, United States). We also used anti-pS6(S235/S236) (#2211), -S6 (#2217), -tuberin (#3612), and -ß-actin (#4970) antibodies from Cell Signaling Technology (Danvers, United States) and anti-α-tubulin (B-5-1-2, #T5168) from Sigma.

Transfection of siRNA was performed with Lipofectamine RNAi MAX (Thermo Fisher Scientific) according to the manufacturer's protocol. Cells were treated with 10 nM siRNA for the Hspb1 gene (5'-CGGAGGAGCUCACAGUGAATT-3' and 5'-UUCACUGUGAGCUCCUCCGGA-3') (36) or control RNA (Thermo Fisher Scientific, #4390843) for 48-72 h. To analyze cell proliferation, rapamycin treatment was started one day after siRNA transfection to avoid severe toxicity.

The full-length cDNA of mouse Hspb1 was amplified using primers mHsp27-F, 5'-AAGAATTCCCAGTGCTTCTAGATCCTCA-3' (forward), and mHsp27-R, 5'-AACTCGAGGCAATGGCTATGGGAGATAG-3' (reverse). The amplified fragment was digested with EcoRI and XhoI and subcloned into the pBabe-puro vector (pBabe-puro was a gift from Hartmut Land & Jay Morgenstern & Bob Weinberg (Addgene plasmid #1764 ; http://n2t.net/addgene:1764 ; RRID:Addgene_1764)) (37). The vector had been modified to contain the XhoI recognition sequence by introducing synthetic oligonucleotides to the cloning site. The resulting Hspb1 expression vector was designated as pBabe-mHSP27. To generate cDNA for the Ser86-to-Asp86 (S86D) Hspb1 mutant, PCRs were carried out with specific primers: mHsp27-F and H27ASPR1, 5'-ACCCCGCTGTCGAGCTGT-3' (reverse), H27ASPF1, 5'-ACAGCTCGACAGCGGGGT-3' (forward), and mHsp27-R, using pBabe-mHSP27 as a template. Then, the two amplified fragments were mixed and a second PCR was performed using mHsp27-F and mHsp27-R to amplify the full-length cDNA. Similarly, cDNA for the Ser86-to-Ala86 (S86A) Hspb1 mutant (pBabe-mHsp27-S86A) was amplified using the following primers: mHsp27F, H27ALAR1, 5'-ACCCCGCTTGCGAGCTGT-3' (reverse), H27ALAF1, 5'-ACAGCTCGCAAGCGGGGT-3' (forward), and mHsp27R. The resulting full length mutant cDNA fragments were cloned into the pBabe-puro vector to generate pBabe-mHSP27-S86D and -S86A. The accuracy of the cDNA sequence was checked by the dideoxy-sequencing method using an ABI 310 sequencer (Thermo Fisher Scientific).

Plat-E cells (pre-cultured without puromycin or blasticidin) were transfected with the vector for wild-type, S86A and S86D Hspb1, or empty vector using FuGENE6 transfection reagent (Promega, Wisconsin, United States) according to the manufacturer's protocol. Next, 48 h after transfection, we collected the culture media which was filtered through a 0.45-µm filter. Then, we added polybrene to a final concentration of 8 µg/ml. MKOC cells were infected with supernatant and the selection of stably transduced cells with 0.6 µg/ml of puromycin was started 48 h after infection. Established cell lines were maintained in the presence of puromycin in the medium.

Statistical analyses were performed with Microsoft Excel software (Microsoft, Redmond, United States), and Statistical analysis program for pharmaceutical data (Osaka university, Osaka, Japan; http://www.gen-info.osaka-u.ac.jp/MEPHAS/tukey-e.html). The significance of difference was examined by unpaired Student's t-test for two groups comparison, and one-way ANOVA followed by Tukey's post hoc test for multiple groups comparison. A p value less than 0.05 was considered statistically significant.

To analyze the levels of Hspb1 protein, we treated MKOC cells with rapamycin (20 nM) for 24 and 48 h and then performed western blotting. Rapamycin suppressed the proliferation of Tsc2-deficient cells by approximately 35% (P<0.001) (Fig. 1A). In this condition, we found that the amount of Hspb1 protein was increased by 24 h rapamycin treatment (Fig. 1B). The phosphorylation of Hspb1 on Ser86 (corresponding to Ser82 in human HSPB1), the main phosphorylation site that regulates oligomerization, was also upregulated, concomitant with an increase in the total amount of protein (Fig. 1B). Furthermore, rapamycin treatment did not result in the increased expression of HSP70 and Hsp90 (Fig. 1C), thus revealing that each HSP responded differently to rapamycin. To ascertain whether these phenomena occurred generally in cultured cells, we investigated the effects of rapamycin on HSP expression in routine HeLa and COS7 cells. No induction of Hspb1 expression was observed in either HeLa or COS7 cells (Fig. 2). These results indicated that the suppression of mTORC1 by rapamycin causes the upregulation of Hspb1 expression in Tsc2-deficient cells.

To investigate whether Hspb1 exerted a positive or negative role in cell proliferation under rapamycin treatment, we used RNAi to suppress the expression of Hspb1. The efficacy of siRNA for the Hspb1 gene was confirmed by western blotting analysis (Fig. 3A). Hspb1 siRNA treatment significantly inhibited the proliferation of MKOC cells under normal culture condition (P<0.05) (Fig. 3B). Under the rapamycin treatment, however, the extent of inhibition was similar when compared between control RNA and Hspb1 siRNA conditions (P<0.05 between DMSO and rapamycin without siRNAs; no significance between control and Hspb1 siRNA under the rapamycin-treated condition) (Fig. 3B), thus suggesting that rapamycin-induced growth regulation may involve the suppression of Hspb1-related pathways.

Next, we analyzed the effects of the forced expression of Hspb1 on cell proliferation. We established MKOC cells stably expressing Hspb1 and control cells (Fig. 4A). When cultured without rapamycin for 48 h, the proliferation of HSP27-expressing cells was promoted when compared with control cells (P<0.05) (Fig. 4B). Under the action of rapamycin, there was also proliferation promoting activity in the wild-type Hspb1 (P<0.05) (Fig. 4B). These results suggested that the function of Hspb1 promoted proliferation in Tsc2-deficient cells even in the presence of rapamycin.

We also assayed the effect of Ser86 phosphorylation in Hspb1 upon cell proliferation by the forced expression of a phosphorylation-mimetic S86D mutant (D-mutant) and resistant S86A mutant (A-mutant) Hspb1 in MKOC cells (Fig. 4A). Western blotting revealed a reduction in pHspb1 antibody binding in D-mutant and A-mutant cases, thus suggesting that site-directed mutagenesis had been successfully accomplished. In the proliferation assays, both with and without rapamycin treatment, there was a reduction of proliferation-promoting activity in the A-mutant case (P<0.05 vs. wild-type case both with and without rapamycin) when compared with the wild-type case, thus suggesting that phosphorylation on S86 might be a positive regulator of proliferation-promoting activity in Hspb1 (Fig. 4A and B). However, there was no obvious difference in cell proliferation when compared between the A- and D-mutant (Fig. 4A and B). From these observations, it was difficult to conclude that Ser86 phosphorylation on Hspb1 affects cell proliferation.