Journal: Cells

Article Title: Silencing Heat Shock Protein 27 Inhibits the Progression and Metastasis of Colorectal Cancer (CRC) by Maintaining the Stability of Stromal Interaction Molecule 1 (STIM1) Proteins

doi: 10.3390/cells7120262

Figure Lengend Snippet: Primer sequences used QPCR analysis with gene name.

Article Snippet: For determination of the association of HSP27 and STIM1 expression levels, two consecutive formalin-fixed paraffin-embedded tissue microarray sections purchased from SuperBioChips Laboratories (catalog no. CDA3, Seoul, Korea) were stained with HSP27 and STIM1 (catalog no. HPA012123, 1:200, Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) by immunohistochemistry using the aforementioned protocol.

Techniques: Sequencing

Journal: Cells

Article Title: Silencing Heat Shock Protein 27 Inhibits the Progression and Metastasis of Colorectal Cancer (CRC) by Maintaining the Stability of Stromal Interaction Molecule 1 (STIM1) Proteins

doi: 10.3390/cells7120262

Figure Lengend Snippet: HSP27 increased the STIM1 protein levels in CRC cells. ( a ) QPCR was used to determine the STIM1 mRNA levels, and the levels were normalized to the GAPDH level. ( b , c and d ) Protein extracts prepared from the indicated cell lines were subjected to western blot analysis for STIM1 or Orai1. GAPDH was used as an internal control. Data are representative of three independent experiments. Values are the mean ± SD of three independent experiments. ** p < 0.01.

Article Snippet: For determination of the association of HSP27 and STIM1 expression levels, two consecutive formalin-fixed paraffin-embedded tissue microarray sections purchased from SuperBioChips Laboratories (catalog no. CDA3, Seoul, Korea) were stained with HSP27 and STIM1 (catalog no. HPA012123, 1:200, Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) by immunohistochemistry using the aforementioned protocol.

Techniques: Western Blot, Control

Journal: Cells

Article Title: Silencing Heat Shock Protein 27 Inhibits the Progression and Metastasis of Colorectal Cancer (CRC) by Maintaining the Stability of Stromal Interaction Molecule 1 (STIM1) Proteins

doi: 10.3390/cells7120262

Figure Lengend Snippet: HSP27 prevented STIM1 degradation by directly binding to STIM1. ( a , b ) Scrambled control or HSP27KD cells were treated with cycloheximide (CHX), MG132 or PYR-41. Whole cell lysates were immunoblotted for STIM1 and HSP27. GAPDH was used as an internal control. ( c ) The possibility of direct protein-protein interactions was examined by immunoprecipitation (IP). All the experiments were independently performed at least 3 times.

Article Snippet: For determination of the association of HSP27 and STIM1 expression levels, two consecutive formalin-fixed paraffin-embedded tissue microarray sections purchased from SuperBioChips Laboratories (catalog no. CDA3, Seoul, Korea) were stained with HSP27 and STIM1 (catalog no. HPA012123, 1:200, Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) by immunohistochemistry using the aforementioned protocol.

Techniques: Binding Assay, Control, Protein-Protein interactions, Immunoprecipitation

Journal: Cells

Article Title: Silencing Heat Shock Protein 27 Inhibits the Progression and Metastasis of Colorectal Cancer (CRC) by Maintaining the Stability of Stromal Interaction Molecule 1 (STIM1) Proteins

doi: 10.3390/cells7120262

Figure Lengend Snippet: HSP27 promoted STIM1 oligomerization in DLD-1 cells. Scrambled control or HSP27KD DLD cells were seeded into coverslips and then treated with vehicle or TG for 15 min. Cells were fixed, and the distribution of STIM1 was detected by immunocytochemical staining. Confocal microscopy was applied to observe the distribution of STIM1 (green). Nuclei were identified by DAPI. All experiments were independently performed at least 3 times.

Article Snippet: For determination of the association of HSP27 and STIM1 expression levels, two consecutive formalin-fixed paraffin-embedded tissue microarray sections purchased from SuperBioChips Laboratories (catalog no. CDA3, Seoul, Korea) were stained with HSP27 and STIM1 (catalog no. HPA012123, 1:200, Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) by immunohistochemistry using the aforementioned protocol.

Techniques: Control, Staining, Confocal Microscopy

Journal: Cells

Article Title: Silencing Heat Shock Protein 27 Inhibits the Progression and Metastasis of Colorectal Cancer (CRC) by Maintaining the Stability of Stromal Interaction Molecule 1 (STIM1) Proteins

doi: 10.3390/cells7120262

Figure Lengend Snippet: The association between HSP27 and STIM1 expression. ( a ) The scatterplot of H-scores of HSP27 and the corresponding STIM1 expression displayed a positive correlation (correlation coefficient = 0.416, p = 0.003). ( b ) A case of colon adenocarcinoma with a high HSP27 H-score displayed a high STIM1 H-score. ( c ) A case of colon adenocarcinoma with a low HSP27 H-score displayed a low STIM1 H-score.

Article Snippet: For determination of the association of HSP27 and STIM1 expression levels, two consecutive formalin-fixed paraffin-embedded tissue microarray sections purchased from SuperBioChips Laboratories (catalog no. CDA3, Seoul, Korea) were stained with HSP27 and STIM1 (catalog no. HPA012123, 1:200, Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) by immunohistochemistry using the aforementioned protocol.

Techniques: Expressing

Journal: Cells

Article Title: Silencing Heat Shock Protein 27 Inhibits the Progression and Metastasis of Colorectal Cancer (CRC) by Maintaining the Stability of Stromal Interaction Molecule 1 (STIM1) Proteins

doi: 10.3390/cells7120262

Figure Lengend Snippet: The proposed model for the role of HSP27 in CRC progression. HSP27 may interact with STIM1 to maintain the stability of STIM1. Silencing HSP27 caused a reduction of STIM1 to regulate the growth activity and metastasis ability on CRC.

Article Snippet: For determination of the association of HSP27 and STIM1 expression levels, two consecutive formalin-fixed paraffin-embedded tissue microarray sections purchased from SuperBioChips Laboratories (catalog no. CDA3, Seoul, Korea) were stained with HSP27 and STIM1 (catalog no. HPA012123, 1:200, Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) by immunohistochemistry using the aforementioned protocol.

Techniques: Activity Assay

Journal: The Journal of Investigative Dermatology

Article Title: Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca 2+ i Mobilization and NFAT2 Activation

doi: 10.1038/jid.2012.370

Figure Lengend Snippet: Lysophosphatidic acid (LPA)-induced Ca 2+ entry requires sufficient levels of STIM1 and functional Orai1. ( a – c ) Relative efficiency of small interfering RNA (siRNA)-mediated STIM1 silencing compared to scrambled (scr) control by quantitative PCR and western blotting. ( e ) Fura-PE3-based Ca 2+ imaging showed impaired LPA-induced Ca 2+ entry in STIM1-knockdown cells. ( e , f ) Efficient transfection of Orai1-encoding plasmids demonstrated by western blotting for myc-tag. ( g ) LPA-induced Ca 2+ entry was found to be blocked in Orai1 R91W -expressing cells. ( h ) Keratinocytes were cotransfected with Orai1, nuclear factor of activated T cell (NFAT)-directed, and Renilla luciferase plasmids. Cells were then treated with LPA and 1.2 mℳ Ca 2+ and assessed for NFAT transcriptional activity. Overexpression of Orai1 R91W significantly impaired NFAT activation. Data represent mean±SEM; n ⩾3 unless otherwise stated, * P <0.05, ** P <0.01, *** P <0.001, analysis of variance.

Article Snippet: Knockdown of STIM1 and NFAT2 was verified by real-time PCR and western blotting using standard techniques ( Hampton et al. , 2012 ) and anti-STIM1 antibody (clone 5A2; Abnova, Taipei, Taiwan) or anti-NFAT2 antibody (a kind gift from Dr Nancy Rice) ( Hampton et al. , 2012 ), respectively.

Techniques: Functional Assay, Small Interfering RNA, Control, Real-time Polymerase Chain Reaction, Western Blot, Imaging, Knockdown, Transfection, Expressing, Luciferase, Activity Assay, Over Expression, Activation Assay

Journal: The Journal of Investigative Dermatology

Article Title: Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca 2+ i Mobilization and NFAT2 Activation

doi: 10.1038/jid.2012.370

Figure Lengend Snippet: Lysophosphatidic acid (LPA)-induced keratinocyte migration requires STIM1, calcineurin activity, and sufficient levels of nuclear factor of activated T cell 2 (NFAT2). ( a – d , h , i ) Keratinocyte cultures were subjected to three-dimensional (3D) chemotactic migration assays, which were performed in medium containing 60 μℳ or 1.2 mℳ Ca 2+ . The average numbers of migrated cells are represented as bar graphs±SEM. Keratinocytes were treated with scrambled (scr) or STIM1-targeted small interfering RNA (siRNA) ( a , b ) or NFAT2-targeted siRNA ( h , i ) for 24 hours prior to 3D chemotactic migration assays. In both Ca 2+ conditions, 10 μℳ LPA significantly increased migration rates. RNA interference (RNAi)-mediated knockdown of STIM1 and NFAT2 resulted in a significant impairment of LPA-induced keratinocyte motility ( a , b , h , i , *** P <0.001, ** P <0.01, two-way analysis of variance (ANOVA)). ( c , d ) Preincubation of cells with 1 μℳ cyclosporin A significantly impaired LPA-induced migration ( n ⩾3, ** P <0.01, * P <0.05, two-way ANOVA). ( e – g ) Keratinocytes were transfected using scr or NFAT2-targeted siRNA. After 24 hours, cells were subjected to ( e ) real-time PCR analysis of NFAT2 expression ( n =6), ( f ) western blot analysis, and ( g ) densitometry ( n =3). RNAi-mediated knockdown of NFAT2 is significant (* P <0.05, t -test). Bar=50 μm.

Article Snippet: Knockdown of STIM1 and NFAT2 was verified by real-time PCR and western blotting using standard techniques ( Hampton et al. , 2012 ) and anti-STIM1 antibody (clone 5A2; Abnova, Taipei, Taiwan) or anti-NFAT2 antibody (a kind gift from Dr Nancy Rice) ( Hampton et al. , 2012 ), respectively.

Techniques: Migration, Activity Assay, Small Interfering RNA, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: The Journal of Investigative Dermatology

Article Title: Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca 2+ i Mobilization and NFAT2 Activation

doi: 10.1038/jid.2012.370

Figure Lengend Snippet: Proposed mechanism of lysophosphatidic acid (LPA)-induced Ca 2+ signaling involved in promoting keratinocyte migration. On the basis of our results, this model proposes that in physiological conditions, LPA promotes migration of keratinocytes through Ca 2+ mobilization mediated by STIM1 and Orai1 and subsequent activation of calcineurin and nuclear factor of activated T cell 2 (NFAT2). ER, endoplasmic reticulum.

Article Snippet: Knockdown of STIM1 and NFAT2 was verified by real-time PCR and western blotting using standard techniques ( Hampton et al. , 2012 ) and anti-STIM1 antibody (clone 5A2; Abnova, Taipei, Taiwan) or anti-NFAT2 antibody (a kind gift from Dr Nancy Rice) ( Hampton et al. , 2012 ), respectively.

Techniques: Migration, Activation Assay