Identification of Suz-12 as a functional target of miR-200b in CSCs.

<p>(<b>A</b>) qRT-PCR detection of relative Suz-12 mRNA expression in the indicated cells. Data were normalized to GAPDH RNA and determined relative to the corresponding parental-cell groups. (<b>B</b>) The protein levels of Suz-12 as measured by Western blotting in the indicated cells. GAPDH was used as an internal control. (<b>C</b>) qRT-PCR detection of Suz-12 mRNA expression in CSCs after transfection of the indicated vectors. (<b>D</b>) Western blotting detection of Suz-12 protein expression in CSCs after transfection with the indicated vectors. GAPDH was used as an internal control. (<b>E</b>) Luciferase activity of the CSCs (SPC-A1/DTX) co-transfected with pcDNA/miR-200b (or pcDNA/miR-NC) or pLUC/Suz-12/3′-UTR-wt (or pLUC/Suz-12/3′-UTR-mut). Data were mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Properties of CSCs obtained from docetaxel-resistant human lung adenocarcinoma cells (SPC-A1/DTX and H1299/DTX).

<p>(A₁, A₂, A₃) Percentage of CD133⁺/CD326⁺ CSCs as analyzed by Flow cytometry at the indicated time after cultivating CD133⁺/CD326⁺ CSCs (acquired by sorting SPC-A1/DTX and H1299/DTX cells, respectively) under differentiation conditions. (B) Mammosphere forming ability of the indicated cells (1000 cells) after 7 days under CSC-cultivating conditions. (C) qRT-PCR detection of relative mRNA levels of the CSC-related markers in the indicated cells. Data was calculated with the 2^−△△Ct method using GAPDH RNA as the reference gene. The expression level was measured relative to the fold change of the parental cells groups that were defined as 1.0. (D₁, D₂) The protein levels of the CSC-related markers in the indicated cells. GAPDH was used as an internal control. (E) Tumor incidence in nude mice that were subcutaneously injected with the indicated cells. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Silencing of Suz-12 inhibits CSCs growth and tumorigenicity and reverses chemoresistance of CSCs.

<p>(<b>A</b>) The protein level of Suz-12 in docetaxel-resistant LAD cells after transfection with the indicated sh-RNA vectors (sh-RNA-Suz12#1, sh-RNA-Suz12#2, and sh-RNA-Suz12#3). (<b>B</b>) Mammosphere forming ability of docetaxel-resistant LAD cells (1000 cells) after transfection of the indicated vectors. (<b>C</b>) Flow cytometry analysis of percentage of CD133⁺/CD326⁺ CSCs as analyzed by in docetaxel-resistant LAD cells after transfection of the indicated vectors. (<b>D</b>) The protein level of Suz-12 in CSCs after transfection with the sh-RNA-control or sh-RNA-Suz12#3 vectors. (<b>E</b>) CCK-8 analysis of cell viability of the CSCs transfected with the indicated vectors at the indicated time. (<b>F</b>) Effect of Suz-12 inhibition on <i>in vivo</i> tumorigenicity of the CSCs from SPC-A1/DTX cells. (<b>G</b>) Effect of Suz-12 inhibition on chemoresistance of CSCs. Cell viability was measured by CCK-8 assay. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

MiR-200b has effects on CSCs by regulating Suz-12/E-cadherin.

<p>(<b>A</b>) Western blotting detection of E-cadherin protein expression in the indicated cells transfected with the indicated vectors. (<b>B</b>) qRT-PCR detection of E-cadherin mRNA expression as measured by in the CSCs transfected with the indicated vectors. (<b>C</b>) Western blotting detection of E-cadherin protein expression in the CSCs transfected with the indicated vectors. (<b>D</b>) and (<b>E</b>) Suz-12 overexpression rescued the increased level of both mRNA and protein expression of E-cadherin in CSCs (SPC-A1/DTX) mediated by miR-200b upregulation, while silencing of Suz-12 rescued the decreased expression of mRNA and protein of E-cadherin in CSCs (SPC-A1/DTX) induced by miR-200b repression. (<b>F</b>) qRT-PCR detection of miR-200b and Suz-12 mRNA expression at the indicated time after plating CD133⁺/CD326⁺ CSCs under differentiation conditions. (<b>G₁, G₂</b>) The protein levels of Suz-12 and E-cadherin at the indicated time after cultivating CD133⁺/CD326⁺ CSCs under differentiation conditions. (<b>H</b>) MiR-200b upregulation decreased the amount of Suz-12 binding to the promoter of E-cadherin in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. (<b>I</b>) MiR-200b overexpression reduced trimethylation of histone H3-lysine-27 (H3-K27) at E-cadherin promoter in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01; ^#<i>p</i><0.05, ^##<i>p</i><0.01, compared with corresponding 0 day groups.</p

HDAC1 repression or miR-200b overexpression reduces tumorigenicity and reverses chemoresistance of CSCs <i>in vivo</i>.

<p>(<b>A</b>) Tumorigenicity in nude mice subcutaneously injected with CSCs from SPC-A1/DTX cells (5000 cells/mouse, n = 5) that were stably transfected with the indicated vectors previously. (<b>B</b>) Tumor volume in nude mice injected with H1299/DTX cells that were stably transfected with the indicated vectors and were combined with DTX treatment. Data were presented as mean ± SD. (<b>C</b>) The protein level of E-cadherin and Suz-12 in tumors of the indicated groups that were provided at 5 weeks after the inoculation. GAPDH was used as an internal control. (<b>D</b>) Flow cytometry detection of percentage of CD133⁺/CD326⁺ CSCs in tumors of the indicated groups. (<b>E</b>) The mRNA level of miR-200b and Suz-12 in tumors of the indicated groups. Data were normalized to U6 RNA and GAPDH, respectively. Data were presented as mean ± SD of at least three independent experiments. **<i>p</i><0.01.</p