Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA, but not RALB, promotes metastatic growth in spontaneous and experimental models of TNBC lung metastasis. a Representative images (top) and quantitation (bottom) of lungs harvested from mice bearing MDA-MB-231 Ctrl ( n = 5), RALA-KO ( n = 6), or RALB-KO ( n = 6) tumors. Lungs were harvested when early removal criteria (ERC) was reached for each respective cohort. Scale bar is 100μm. b Representative H&E images and quantification of lung metastases arising in mice bearing orthotopic MVT1 shCtrl ( n = 6) or shRALA ( n = 6) mammary tumors. Lung metastasis were categorized as nodules, foci, or emboli. Representative images of each category are shown. Scale bars are 50μm except for the representative foci image which is 20μm. All mice were sacrificed when the shCtrl group met ERC. c Experimental metastasis assay. Representative IVIS images and quantification of total flux in the lungs of mice following tail vein inoculation of luciferase-tagged MDA-MB-231 shCtrl ( n = 15) or shRALA ( n = 10) cells over 24 days. e Extravasation and early colonization assay. Representative IVIS images and quantification of total flux in the lungs of mice following tail vein inoculation of luciferase-tagged MDA-MB-231 shCtrl ( n = 13) or shRALA ( n = 11) cells over 96 h. Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: Quantitation Assay, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: BQU57 blocks TNBC growth in vitro and in vivo. a Western blot and quantification of GTP-bound RALB in MDA-MB-231 cells after BQU57 (50μM) or DMSO treatment for 24 h. b Quantification of MDA-MB-231 and MVT1 cell viability upon treatment with varying doses of BQU57 for 72 h. c Quantification of MDA-MB-231 and MVT1 growth in low adhesion (GILA) conditions upon treatment with varying doses of BQU57 for 5 days. d Quantification of MDA-MB-231 orthotopic mammary tumor volume over time [ n = 9 DMSO; n = 10 BQU57 treatment (50mg/kg by i.p. injection, M-F)]. e Representative H&E images (left) and quantification (right) of spontaneous lung metastases in MDA-MB-231 tumor bearing mice following treatment (arrows point to metastatic lesions). Scale bars = 500μm. f Quantification of subcutaneous TNBC PDX tumor volume over time. [ n = 9 DMSO; n = 9 BQU57 treatment (50mg/kg, M-F)]. g Quantification of MDA-MB-231 cell viability upon treatment with BQU57 (100μM) or DMSO in combination with various doses of paclitaxel for 72 h. h RALA expression, but not RALB expression, is predictive of BC patient response to chemotherapy. Data from ROC Plotter. Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vitro, In Vivo, Western Blot, Injection, Expressing

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA, not RALB, is pro-tumorigenic in preclinical in vivo models of TNBC utilizing CRISPR mediated knockout of RALA/B. a Western blots demonstrating RALA and RALB expression in MDA-MB-231 CRISPR control (Ctrl), RALA CRISPR knockout (RALA-KO), and RALB CRISPR knockout (RALB-KO) cells. b Quantification of RALA and RALB expression in MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells by ImageJ analysis ( n = 3). c Representative images of RALA and RALB immunofluorescence staining in MDA-MB-231 Ctrl, RALA-KO and RALB-KO cells (red = RALA or RALB, blue = DAPI; scale bars = 20μm). d Quantification of MDA-MB-231 Ctrl ( n = 10), RALA-KO ( n = 8), and RALB-KO ( n = 12) orthotopic mammary tumor growth. e Representative images and H-score quantification of Ki67 immunostaining in MDA-MB-231 CRISPR Ctrl, RALA-KO, and RALB-KO orthotopic mammary tumors (scale bar = 40μm). f Representative images and H-score quantification of cleaved caspase 3 (CC3) immunostaining in MDA-MB-231 Ctrl, RALA-KO, and RALB-KO orthotopic mammary tumors (scale bar = 20μm). Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vivo, CRISPR, Knock-Out, Western Blot, Expressing, Control, Immunofluorescence, Staining, Immunostaining

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA, not RALB, is pro-tumorigenic in preclinical in vivo models of TNBC utilizing short hairpin knockdown of RALA/B. a Western blots demonstrating RALA and RALB expression in MDA-MB-231 shRNA control (shCtrl), shRALA, and shRALB cells. b Comparison of MDA-MB-231 shCtrl ( n = 5) and shRALA ( n = 5) orthotopic mammary tumor growth. c Comparison of MDA-MB-231 shCtrl ( n = 7) and shRALB ( n = 8) orthotopic mammary tumor growth. d Representative Ki67 immunostaining images and H-score quantification of MDA-MB-231 shCtrl ( n = 4) and shRALA ( n = 4) orthotopic mammary tumors. e Representative cleaved caspase 3 (CC3) immunostaining images and H-score quantification of MDA-MB-231 shCtrl ( n = 4) and shRALA ( n = 4) orthotopic mammary tumors. f Quantification of Rala mRNA expression in MVT1 shCtrl and shRALA cells by qRT-PCR (left) and growth of orthotopic mammary tumors (right; n =6 per group). Inset shows western blot confirmation of RALA protein expression in MVT1 shCtrl and shRALA cells. Data are presented as mean ± SEM; (*), P < 0.05; scale bars = 20μm

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vivo, Knockdown, Western Blot, Expressing, shRNA, Control, Comparison, Immunostaining, Quantitative RT-PCR

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA supports, while RALB opposes, in vitro measures of aggressive cancer phenotypes in TNBC cell lines. a Representative images and quantification of MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cell migration over 6 h (scale bar = 100μm). b Representative images and quantification of MDA-MB-468 siRNA control (siCtrl), siRALA, and siRALB cell migration over 6 h (scale bar = 100μm). c Representative images and quantification of MVT1 shCtrl and shRALA cell migration over 6 h (scale bar = 200μm). d Representative images and quantification of MDA-MD-231 shCtrl and shRALA scratch assay over 6 h (scale bar = 500μm). e Representative images and quantification of MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cell invasion through matrigel coated transwell inserts over 24 h (scale bar = 200μm). f Representative images and quantification of MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cell invasion as three dimensional spheroids over 5 days (scale bar = 400μm). g Quantification of viability for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells ± siCtrl or siRALA/B transient knockdown over 72 h. h Quantification of viability for MDA-MB-468 siCtrl, siRALA and siRALB cells over 72 h. i Quantification of BrdU incorporation for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells over 72 h. j Quantification of Annexin V positivity for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells. k Quantification of growth in low adhesion (GILA) conditions for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells ± siCtrl or siRALA/B transient knockdown over 5 days. h Quantification of growth in low adhesion (GILA) for MDA-MB-468 siCtrl, siRALA and siRALB cells over 5 days. Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vitro, Migration, Control, Wound Healing Assay, Knockdown, BrdU Incorporation Assay

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA is elevated in TNBC and is prognostic of overall survival in ER-negative disease. a Western blots demonstrating RALA and RALB expression in a panel of BC cell lines denoted by molecular subtype. b RALA and RALB mRNA expression in a panel of 46 BC cell lines categorized according to molecular subtype. Data from the Broad Institute Cancer Cell Line Encyclopedia (CCLE), (*) P < 0.05. c Box and whisker plots of RALA and RALB expression in normal breast tissue ( n = 61), BC subtypes other than TNBC ( n = 300) , and TNBC ( n = 49). Data from TCGA Research Network. (*) P < 0.05. d Box and whisker plots of RALA and RALB expression in normal breast tissue ( n = 144), BC subtypes other than TNBC ( n = 1725), and TNBC ( n = 250). Data from METABRIC. (*) P < 0.05. e Left: Kaplan-Meier analysis segregates all BC patients by RALA (upper quartile vs lower three-quartiles) and RALB (spilt along the median) expression in METABRIC and TCGA datasets wherein high RALA is prognostic of worse disease specific survival (DSS) in the METABRIC ( P = 0.0079) and overall survival in the TCGA ( P = 0.0003) cohorts and low RALB is prognostic of worse DSS in the METABRIC cohort ( P = 0.0311), but is not prognostic in the TCGA cohort ( P = 0.9477). Right: Within the TNBC population of the METABRIC cohort, RALA (upper tenth percentile vs lower 90th percentail) is prognostic of worse DSS TNBC patients ( P = 0.0544) while RALB (spilt along the median) is not prognostic ( P = 0.9297). Significance determined by log-rank. f Top: Representative high and low RALA immunostaining of BC patient samples. Bottom: Kaplan-Meier analysis segregating all BC patients ( P = 0.0417) and TNBC patients ( P = 0.0440) by RALA H-score (upper tertile vs lower two tertiles) where high RALA is prognostic of worse overall survival (Scale bars = 60μm)

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Whisker Assay, Immunostaining

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 4: Loss of Lats and YAP Phosphorylation, Increased TAZ Levels with PCBP2 652

Article Snippet: TAZ and YAP were depleted using siRNA from Origene.

Techniques: Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 7: Requirement of YAP and TAZ for PCBP2-mediated Changes in Transcription. 701

Article Snippet: TAZ and YAP were depleted using siRNA from Origene.

Techniques:

Journal: Scientific Reports

Article Title: Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

doi: 10.1038/s41598-020-80848-3

Figure Lengend Snippet: Gata4, Mef2c, and Tbx5 expression in the beating and non-beating groups. ( a ) Left three panels, gene expression levels in the beating and non-beating group. Right panel, gene expression levels in the beating and non-beating group relative to that of β-actin. ( b ) Representative immunofluorescence images of a cluster of round cells expressing Gata4 and Mef2c on day 14. ( c ) Phase-contrast and immunofluorescence images of beating cells expressing cardiac troponin T (cTnT) and Mef2c on day 28. The arrowhead indicates a beating cell. Scale bar, 100 µm. DAPI, 4′,6-diamidino-2-phenylindole, ** p < 0.01; **** p < 0.0001 by t -test or ANOVA and Tukey’s post hoc test. ( d ) The frequency of round cells expressing Gata4 and Mef2c (10 round cells were examined per experiment, n = 13). ( e ) The frequency of beating cells expressing Mef2c and cTnT (15 beating cells were analysed per experiment, n = 4).

Article Snippet: For silencing of mouse Mef2c, LentiORF lentiviral particles of mouse Mef2c-specific short hairpin RNA (TL511428V, Origene) or scrambled control were used.

Techniques: Expressing, Gene Expression, Immunofluorescence

Journal: Scientific Reports

Article Title: Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

doi: 10.1038/s41598-020-80848-3

Figure Lengend Snippet: Overexpression of SVF with Mef2c augments the number of round cells and their transdifferentiation into CMs. ( a – d ) Phase-contrast images of SVF on day 28 after transfection on day 5 ( a , control; b , Gata4; c , Mef2c; d , Tbx5). Arrowheads indicate round cells; lv, lentivirus vector. ( e ) mRNA level of Mef2c, determined by qRT-PCR, on day 28 in SVFs transfected with the control vector and Mef2c-lv (left), or scrambled shRNA and shMef2c-lv (right), on day 5, respectively. shRNA, short hairpin RNA; shMef2c, shRNA targeting Mef2c. ( f ) Number of round cells on day 14 after introducing SVF with none (none) or the control, Gata4, Mef2c, shMef2c, or Tbx5 lentivirus vector. ( g ) mRNA level of cardiac troponin T by qRT-PCR on day 28 in SVF transfected with the control, Gata4, Mef2c, shMef2c, or Tbx5. ( h ) Relationship between the mRNA levels of Mef2c and cardiac troponin T in the SVF transduced with Mef2c. R, Pearson’s product-moment correlation coefficient. The linear interpolation formula is displayed. Scale bar = 100 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001 by ANOVA and Tukey’s post hoc test.

Article Snippet: For silencing of mouse Mef2c, LentiORF lentiviral particles of mouse Mef2c-specific short hairpin RNA (TL511428V, Origene) or scrambled control were used.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, shRNA, Transduction

Journal: Molecular medicine reports

Article Title: PLAC1 is an independent predictor of poor survival, and promotes cell proliferation and invasion in cervical cancer.

doi: 10.3892/mmr.2021.12440

Figure Lengend Snippet: Figure 1. PLAC1 expression is significantly upregulated in CC and high PLAC1 expression is associated with poor survival. (A) PLAC1 expression was upregu lated in CC tissues compared with in normal cervical tissues, as revealed using TCGA‑CESC dataset. (B) PLAC1 expression was upregulated in cervical squamous cell carcinoma tissues compared with that in normal cervical tissues, as revealed using TCGA‑CESC dataset. In other pathological types of CC, (C) endocervical mucinous adenocarcinoma, (D) adenosquamous carcinoma and (E) cervical adenocarcinoma, PLAC1 expression was upregulated, but did not reach a statistically significant level. Data are presented as the mean ± SEM. *P<0.05, **P<0.01. (F) Kaplan‑Meier survival analysis showed that CC cases with PLAC1‑high expression had a worse prognosis than those with PLAC1‑low expression (P=0.0296). CC, cervical cancer; TCGA‑CESC, The Cancer Genome Atlas Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; PLAC1, placenta‑specific protein 1; ns, not significant; TPM, transcripts per million.

Article Snippet: Lentivirus harboring two short hairpin RNAs (shRNAs) targeting PLAC1 gene (PLAC1 shRNA1 and PLAC1 shRNA2) or PLAC1 cDNA were designed and produced by OriGene Technologies, Inc. shRNA targeting firefly luciferase (shLuc) was used as a negative control for RNA silencing, and empty vector was used as a negative control for cDNA overexpression.

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: PLAC1 is an independent predictor of poor survival, and promotes cell proliferation and invasion in cervical cancer.

doi: 10.3892/mmr.2021.12440

Figure Lengend Snippet: Figure 2. Gene set enrichment analysis. (A) ‘mTORC1 signaling’, (B) ‘interferon α response’ and (C) ‘hypoxia’ were differentially enriched in the ‘high‑PLAC1’ expression phenotype. (D) Heat map of the top 50 features for each phenotype. PLAC1, placenta‑specific protein 1; mTORC1, mTOR complex 1.

Article Snippet: Lentivirus harboring two short hairpin RNAs (shRNAs) targeting PLAC1 gene (PLAC1 shRNA1 and PLAC1 shRNA2) or PLAC1 cDNA were designed and produced by OriGene Technologies, Inc. shRNA targeting firefly luciferase (shLuc) was used as a negative control for RNA silencing, and empty vector was used as a negative control for cDNA overexpression.

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: PLAC1 is an independent predictor of poor survival, and promotes cell proliferation and invasion in cervical cancer.

doi: 10.3892/mmr.2021.12440

Figure Lengend Snippet: Figure 3. Immunohistochemical evaluation of PLAC1 in (A) normal exocervix, (B) cervical squamous cell carcinoma, (C) cervical adenocarcinoma (D) cer vical adenosquamous carcinoma, (E) small cell neuroendocrine cervical cancer and (F) high‑grade squamous intraepithelial lesions. Black arrows show PLAC1 expression. IHC score values of PLAC1 were significantly higher in (G) CC and (H) HSILs than those in normal tissues. Bars indicate the median and interquartile range. *P<0.05, ***P<0.001. (I) Patients with high expression of PLAC1 had poor overall survival (P=0.0111). Magnification, x200. PLAC1, pla centa‑specific protein 1; IHC, immunohistochemistry.

Article Snippet: Lentivirus harboring two short hairpin RNAs (shRNAs) targeting PLAC1 gene (PLAC1 shRNA1 and PLAC1 shRNA2) or PLAC1 cDNA were designed and produced by OriGene Technologies, Inc. shRNA targeting firefly luciferase (shLuc) was used as a negative control for RNA silencing, and empty vector was used as a negative control for cDNA overexpression.

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry

Journal: Molecular medicine reports

Article Title: PLAC1 is an independent predictor of poor survival, and promotes cell proliferation and invasion in cervical cancer.

doi: 10.3892/mmr.2021.12440

Figure Lengend Snippet: Figure 4. PLAC1 promotes proliferation and motility in CC. (A) Expression of PLAC1 in cervical cancer cell lines CaSki, MS751, C‑33A and HeLa. (B) PLAC1 was upregulated in PLAC1‑overexpressing HeLa cells and downregulated in PLAC1‑silenced MS751 cells. (C) Cell proliferation determined using colony formation assay. (D and E) Cell proliferation measured using EdU incorporation assay. Magnification, x200. (F and G) Cell motility examined using wound healing assay. Magnification, x40. (H and I) Cell motility detected using Transwell assay. Magnification, x200. Data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. PLAC1, placenta‑specific protein 1; sh, short hairpin; EdU, ethynyl‑2'‑deoxyuridine.

Article Snippet: Lentivirus harboring two short hairpin RNAs (shRNAs) targeting PLAC1 gene (PLAC1 shRNA1 and PLAC1 shRNA2) or PLAC1 cDNA were designed and produced by OriGene Technologies, Inc. shRNA targeting firefly luciferase (shLuc) was used as a negative control for RNA silencing, and empty vector was used as a negative control for cDNA overexpression.

Techniques: Expressing, Colony Assay, Wound Healing Assay, Transwell Assay

Journal: Molecular medicine reports

Article Title: PLAC1 is an independent predictor of poor survival, and promotes cell proliferation and invasion in cervical cancer.

doi: 10.3892/mmr.2021.12440

Figure Lengend Snippet: Figure 5. Overexpression of PLAC1 significantly increases the expression of EMT‑associated protein and knockdown of PLAC1 significantly decreases the expression of EMT‑associated protein. (A and B) Overexpression of PLAC1 in HeLa cells significantly increased the expression of Slug, MMP2 and Vimentin, but decreased the expression of E‑cadherin, as detected via western blot analysis. (C and D) Knockdown of PLAC1 in MS751 cells significantly decreased the expression of Slug, Snail, MMP2, and Vimentin but increased the expression of E‑cadherin, as detected via western blot analysis. The results were normalized to the expression of β‑actin. *P<0.05, **P<0.01, ***P<0.001. PLAC1, placenta‑specific protein 1; sh, short hairpin; EMT, epithelial‑mesenchymal transition.

Article Snippet: Lentivirus harboring two short hairpin RNAs (shRNAs) targeting PLAC1 gene (PLAC1 shRNA1 and PLAC1 shRNA2) or PLAC1 cDNA were designed and produced by OriGene Technologies, Inc. shRNA targeting firefly luciferase (shLuc) was used as a negative control for RNA silencing, and empty vector was used as a negative control for cDNA overexpression.

Techniques: Over Expression, Expressing, Knockdown, Western Blot

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: MAGED4B mRNA has low expression in the STAD tissues and is negatively correlated with PFS. ( A ) PFS of STAD samples in the TCGA database; ( B ) Transcript expression level of MAGED4B in the STAD—para-carcinoma clinical samples; ( C ) Protein expression level of MAGED4B in 76 clinical specimens; ( D ) IHC results. **P<0.01, ***P<0.001.

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques: Expressing

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: Relationship Between MAGED4B Expression and Clinical Characteristics in Stomach Adenocarcinoma

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques: Expressing

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: Expressions of MAGED4B in different STAD cell lines.

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques:

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: Construction of cell lines with overexpressed/silent MAGED4B. ( A and B ) qPCR was performed to detect the expression of MAGED4B in the cell lines with overexpressed/silent MAGED4B; ( C and D ) WB was performed to detect the protein expression of MAGED4B in the cell lines with overexpressed/silent MAGED4B; ( E and F ) Statistical results of MAGED4B protein expression the cell lines with overexpressed/silent MAGED4B. *P<0.05, **P<0.01, and ****P<0.0001.

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques: Expressing

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: Overexpression of MAGED4B promotes cell migration, viability and proliferation. ( A and B ) Scratch test was performed to evaluate the migration ability of cell lines with overexpressed/silent MAGED4B; ( C and D ) CCK-8 to detect the cell viability of cell lines with overexpressed/silent MAGED4B; ( E and F ) EDU was performed to evaluate the cell proliferation ability of cell lines with overexpressed/silent MAGED4B. ****P<0.0001.

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques: Over Expression, Migration, CCK-8 Assay

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: MAGED4B inhibits apoptosis of STAD cells and their sensitivity to cisplatin. ( A ) IC 50 of cisplatin in the BGC and HGC-27 cells; ( B ) After treatment with 8 µ g/mL cisplatin for 24 h, the apoptosis of BGC and HGC-27 cells was detected by flow cytometry. ( C ) After treatment with 8 µ g/mL cisplatin for 24 h, the apoptosis of BGC and HGC-27 cells was detected by flow cytometry. ****P<0.0001.

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques: Flow Cytometry

Journal: International Journal of General Medicine

Article Title: MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis

doi: 10.2147/IJGM.S401507

Figure Lengend Snippet: Expressions of related proteins in the cell lines with overexpressed and silent MAGED4B. ( A ) Expressions of related proteins detected by WB; ( B ) Statistics of protein expressions by WB. *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: According to the manufacturer’s instructions, the MAGED4B Human shRNA Lentiviral Particle (TL303375V, OriGene Technologies, Inc., USA) was used to knock out the MAGED4B genes from the HGC-27 cells.

Techniques: