Legend to Supplementary Table 1.
Supplementary Table 1. In silicoanalysis of HDAC4-targeting by miR-29b. Summary of bioinformaticsoftwares predicting the targeting of HDAC4 by miR-29b obtained by mirDIP analysis.
Legend to Supplementary Figures
Supplementary Figure 1. miR-29b targets HDAC4 in SKMM1 cells. A. qRT-PCR analysis of miR-29b expression in SKMM1 cells, 24h after transfection with 200nM miR-29b- or scrambled (NC)- oligonucleotides. B. Dual-luciferase assay of SKMM1 cells co-transfected with firefly luciferase constructs containing the 3’ UTR of HDAC4 or a deletion mutant lacking the predicted miR-29b target site (3’ UTR del) and 200nM of miR-29b or scrambled oligonucleotides (NC) as indicated. The firefly luciferase activity was normalized to renilla luciferase activity. *P<0.01 C. qRT–PCR of HDAC1, HDAC2, HDAC4 and HDAC6 in SKMM1 cells, 24h after transfection with 200nM miR-29b or scrambled oligonucleotides (NC). D. Immunoblot of HDAC4, acetyl-histone H4 and GAPDH in SKMM1 transfected with 200nM miR-29b- or scrambled (NC) oligonucleotides. E. Immunoblot of acetyl--tubulin 24h after transfection of SKMM1 with 200nM miR-29b or scrambled (NC) oligonucleotides. GAPDH was used as a loading control.F.qRT–PCR of miR-29b in SKMM1 cells transduced with the empty vector (carrying GFP) or antagomiR-29b (anti-miR-29b) lentiviral vector. Results are average mRNA expression after normalization with GAPDH and Ct calculations. Immunoblot shows the levels of HDAC4 in GFP- or anti-miR-29b-transduced SKMM1 cells. GAPDH was used as a loading control.*P<0.01
Supplementary Figure 2. miR-29b overexpression and HDAC4-silencingaffectMM cell autophagy. A. CYTO-ID analysis of autophagy performed in KMS11 cells stably transduced with shRNAs targeting HDAC4 or scrambled (SCR) controls. Results are expressed as Mean of Fluorescence Intensity (MFI) as determined by FACS-analysis. *P<0.01.B. Immunoblot analysis of LC3A, LC3B, Beclin-1, TFEB and p62/SQSTM1 in KMS11 cells transfected with 200nM miR-29b or scrambled (NC) synthetic oligonucleotides. C. CYTO-ID analysis of autophagy performed in KMS11 cells transfected with 200nM synthetic miR-29b or NC and then treated for 48h with or without 10M of the autophagy inducer Tamoxifen (TAM). *P<0.01.D.Cell Titer Glo (Promega) survivalassay performed in KMS11 cells stably transduced withshRNAs targeting HDAC4 or SCR controls, and then treated for 24h with the autophagy inhibitor Chloroquine (CQ) at the indicated concentrations. *P<0.01.
Supplementary Figure 3.Analysis of apoptosis and autophagy triggered by SAHA and miR-29b mimics in MM cells. A. Annexin V-staining of KMS11cells transfected with 200nM of NC or miR-29b mimics, and then treated with SAHA for 48h.*P<0.05. B. Immunoblot analysis of phosporylated-mTOR (Ser2448), TFEB, p62/SQSTM1, Beclin-1, LC3B, total and cleaved caspase 3 (CF) in KMS11 and SKMM1 treated for 24h with SAHA at the indicated concentrations. GAPDH or-tubulin were used as loading control.
Supplementary Figure 4.miR-29b inhibition impairs the effects of SAHA on NCI-H929 cell apoptosis and migration. A. Caspase 3/7 activity was determined in NCI-H929 cells stably transduced with a control empty vector (GFP) or antagomiR-29b (anti-miR-29b). Results are expressed as percentage of caspase activity of control cells. *P<0.05. B. Immunoblot of caspase 3 in NCI-H929 transduced with GFP or anti-miR-29b, and then treated with SAHA. GAPDH was used as a loading control. C. Transwell migration assay in NCI-H929 cells transduced with GFP or anti-miR-29b, and then treated with SAHA 0.5M for 24h. *P<0.01.
Supplementary Figure 5. In vivo effects of miR-29b plus SAHA in KMS11 xenografts. Representative picture of mice treated with NC or miR-29b mimics (1mg/Kg), SAHA (20mg/Kg) or combination (COMBO) treatment, obtained by IVIS II imaging system.