plko 1 sox2 3hm  (Addgene inc)

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A–D ) Pluripotency genes expressions analyzed by qPCR in HCC827 ( A ), PDCL#24 ( B ), A549 ( C ), and H1299 cells ( D ) with NFATc2 knockdown or overexpression. ( E ) Effects of stable NFATc2 knock-down, knockout or overexpression on SOX2 expression in respective lung cancer cells by Western blot analysis. ( F–G ) Pluripotency genes expression analyzed by qPCR in HCC827 ( F ), and PDCL#24 cells ( G ) treated with CSA or FK506, respectively, for 24 hr. ( H–I ) Effects of transient knockdown of PPP3R1 on pluripotency gene expressions analyzed by qPCR in HCC827 ( H ) and PDCL#24 cells ( I ). ( J–K ) Effects of transient knockdown of NFATc1 on pluripotency gene expressions analyzed by qPCR in HCC827 ( J ) and PDCL#24 cells ( K ). *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.017

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Knockdown, Over Expression, Knock-Out, Expressing, Western Blot, Control

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A ) Computational prediction of NFAT binding sites (marked in red) on 5’ and 3’ SOX2 regulatory regions (Region 1 to 4). TSS: transcription start site. ( B ) Transcriptional activities of the respective SOX2 regions 1–4 of H441 cells analyzed by luciferase reporter assays. *p<0.05, **p<0.01 versus control by Student’s t-test. Error bars indicate the mean ±SD for at least three independent replicates. ( C–D ) Luciferase reporter activities of mutant or wild-type SOX2 reporters of A549 ( C ), or H1299 cells ( D ), with or without NFATc2 stable overexpression. *p<0.05, **p<0.01, comparison with RFP; # p<0.05, ## p<0.01, wild type versus mutant in RFP-NFATc2 cells by t-test. Error bars indicate the mean ±SD for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.021

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Binding Assay, Luciferase, Control, Mutagenesis, Over Expression, Comparison

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A ) Genome browser view of NFAT binding sites and H3K27Ac marks (lowest panel) on SOX2 regulatory regions (regions 2 and 3 indicated in ) analyzed in A549 cells. ( B ) Transcriptional activities of sites 1–5 by dual luciferase reporter assays in H441 cells. ( C ) Transcriptional activities of the indicated putative NFAT binding sites by respective luciferase reporters in H441 cells with transient NFATc2 over-expression, with or without CSA treatment. ( D ) Effects of site-directed mutagenesis of the indicated putative NFAT binding sequences by respective luciferase reporter assays in H441 cells with transient NFATc2 overexpression. For B-D, *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. ( E ) Alignment of sites 4 and 5 genomic sequences showing highly homologous regions (gray) in different mammalian species, with putative NFAT binding sites highlighted in red. ( F–G ) ChIP–qPCR assays of NFATc2 binding to the indicated SOX2 sites in A549 cells with or without stable NFATc2 overexpression ( F ), or HCC827 cells with or without NFATc2 knockout ( G ). # p<0.05, ## p<0.01 versus IgG control,m **p<0.01 versus vector control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. ( H ) Correlation of immunohistochemical expressions of NFATc2 and SOX2 in 92 moderately to poorly differentiated human lung adenocarcinoma by χ 2 -test. Pearson R, Pearson correlation coefficient. ( I ) Correlation of mRNA levels of SOX2 and NFATc2 in a panel of lung AD cell lines analyzed by q-PCR and Pearson correlation test. ( J ) Expression of NFATc2 and SOX2 in A549 cells with or without NFATc2 overexpression and SOX2 stable knockdown by Western blot. ( K–L ) Effect of SOX2 knockdown on tumorsphere formation ( K ), cell migration and invasion ability ( L ), of A549 cells with NFATc2 overexpression. *p<0.05, **p<0.01 versus control by t-test. Error bars indicate the mean ±SD for at least three independent replicates. ( M ) In vivo tumorigenicity of A549 cells with NFATc2 overexpression and SOX2 knockdown by subcutaneous inoculation of 1 × 10 4 cells in SCID mice. **p<0. 0001 versus control by two-way ANOVA. Error bar indicates the mean ±SD of tumor volumes of six mice. ( N ) Effect of NFATc2 knockdown on SOX2 expression in A549 CR cells analyzed by immunoblot. ( L ) Effect of SOX2 knockdown on cisplatin sensitivity by MTT assay of A549 cells with NFATc2 overexpression. ## p<0.01, versus vector control, **p<0.01 versus RFP-NFATc2_Sh-Ctrl by t-test. Error bar indicates the mean ±SD for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.019 10.7554/eLife.26733.020 Figure 6—source data 1. Statistical analyses for . DOI: http://dx.doi.org/10.7554/eLife.26733.020

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Binding Assay, Luciferase, Over Expression, Mutagenesis, Control, Genomic Sequencing, ChIP-qPCR, Knock-Out, Plasmid Preparation, Immunohistochemical staining, Expressing, Knockdown, Western Blot, Migration, In Vivo, MTT Assay

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A–D ) Effects on ALDH + , CD44 + and ALDH + /CD44 + cell populations by flow cytometry analysis of HCC827 with NFATc2 knockdown ( A ), NFATc2 knockout ( B ), or NFATc2 inhibition by CSA or FK506 ( C ), and of A549 cells with NFATc2 overexpression ( D ). ( E–H ) Effects on ALDH1A1 mRNA expression by qPCR analysis of cancer cells with NFATc2 knockdown ( E ), NFATc2 inhibition by CSA or FK506 ( F ), NFATc2 up-regulation ( G ), or of A549 with induced cisplatin resistance ( H ). ( I ) Effects of SOX2 knockdown on ALDH + , CD44 + and ALDH + /CD44 + cell populations by flow cytometry in A549 with NFATc2-overexpression. ( J ) Expression of SOX2 and ALDH1A1 transcripts in A549 cells with NFATc2 overexpression and SOX2 knockdown. ( K ) Representative images of A549 xenografts with or without NFATc2 overexpression immunohistochemically stained for NFATc2, SOX2 and ALDH1A1, respectively. Scale bars, 50 µm. ( L ) Conserved SOX2 binding sequences (ATTCA) at ALDH1A1 enhancer region by ChIP-seq of PDCL#24 cells, aligned with homologous mammalian sequences and H3K27Ac peaks of A549 cells from published databases. ( M ) Detection of endogenous SOX2 binding to ALDH1A1 sites by ChIP–qPCR analysis in PDCL#24 cells. ( N ) Luciferase reporter activities at sites 1 and 2 of ALDH1A enhancer region by dual luciferase reporter assay in A549 cells with SOX2 overexpression. ( O–P ) Effects of transient ALDH1A1 suppression on A549 with upregulated NFATc2 with respect to invasion and migration ( O ), and cisplatin sensitivity. ## p<0.01, versus RFP_Scramble by t-test. ( P ). ( Q ) Correlation between ALDH1A1 and SOX2 expressions by IHC in human lung adenocarcinomas by χ 2 -test. *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.022 10.7554/eLife.26733.023 Figure 7—source data 1. Statistical analyses for . DOI: http://dx.doi.org/10.7554/eLife.26733.023

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Flow Cytometry, Knockdown, Knock-Out, Inhibition, Over Expression, Expressing, Staining, Binding Assay, ChIP-sequencing, ChIP-qPCR, Luciferase, Reporter Assay, Migration, Control

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A ) ROS levels detected by flow cytometry in A549 and A549 CR cells. ( B–C ) ROS levels in HCC827 cells ( B ) and PDCL#24 cells ( C ) with or without NFATc2 stable knockdown. ( D ) ROS levels in HCC827 cells with or without NFATc2 knockout. ( E–F ) Cisplatin sensitivity expressed as IC 50 by MTT assays of NFATc2-silenced PDCL#24 cells treated with increasing doses of NAC ( E ), or NFATc2-overexpressing A549 cells treated with the oxidizing agent BSO ( F ), respectively. *p<0.05, **p<0.01 versus vector control without REDOX reagents; ## p<0.01 versus the corresponding treatment control by t-tests. Error bar indicates the mean ±S.D. for three independent replicates. ( G–H ) Effects of increasing doses of NAC on tumorsphere formation ability of HCC827 ( G ) cells and PDCL#24 cells ( H ). *p<0.05, **p<0.01 versus corresponding treatment controls, ## p<0.01 versus vector control by t-test. Error bar indicates the mean ±S.D. for three independent replicates. ( I ) Effects of increasing doses of NAC on cell migration and invasion of HCC827 cells with NFATc2 down-regulation by 2 sh-RNA knockdown sequences. ( J–K ) ROS levels in NFATc2-overexpressing A549 cells with stable SOX2 ( J ) or transient ALDH1A1 ( K ) knockdown. *p<0.05, **p<0.01 versus respective control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.028 10.7554/eLife.26733.029 Figure 8—source data 1. Statistical analyses for . DOI: http://dx.doi.org/10.7554/eLife.26733.029

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Flow Cytometry, Knockdown, Knock-Out, Plasmid Preparation, Control, Migration

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: Expressions of total β-catenin, active β-catenin (non-phosphorylated), and phosphorylated β-catenin (p-β-catenin) analyzed by immunoblot in A549 with or without NFATc2 overexpression and SOX2 knockdown. DOI: http://dx.doi.org/10.7554/eLife.26733.027

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Western Blot, Over Expression, Knockdown