Journal: Molecular cancer research : MCR

Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0042

Figure Lengend Snippet: A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

Article Snippet: Rabbit anti-RNF20 antibody (#9425) was purchased from Cell Signaling Technology (Beverly, CA, USA).

Techniques: Western Blot, Biomarker Assay, Flow Cytometry, Expressing, Generated, Isolation

Journal: Molecular cancer research : MCR

Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0042

Figure Lengend Snippet: A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

Article Snippet: Rabbit anti-RNF20 antibody (#9425) was purchased from Cell Signaling Technology (Beverly, CA, USA).

Techniques: Western Blot, Transfection, shRNA, In Vivo

Journal: Journal of Medicine and Life

Article Title: 25-OH Vitamin D blood serum linkage with VDR gene polymorphism (rs2228570) in thyroid pathology patients in the West-Ukrainian population

doi: 10.25122/jml-2021-0101

Figure Lengend Snippet: Demographic, clinical, and biochemical characteristics of study participants.

Article Snippet: 25-OH vitamin D levels in the serum of the patients and healthy individuals were quantified with ELISA using the 25-OH vitamin D Total (Vit D-Direct) Test System ELISA Kit (Monobind Inc.®, United States, Product Code: 9425-300) on the EIA Reader Sirio S (Seac, Italy).

Techniques:

Journal: Journal of Medicine and Life

Article Title: 25-OH Vitamin D blood serum linkage with VDR gene polymorphism (rs2228570) in thyroid pathology patients in the West-Ukrainian population

doi: 10.25122/jml-2021-0101

Figure Lengend Snippet: 25-OH vitamin D levels in groups of patients with different thyroid pathology depending on the rs2228570 genotype.

Article Snippet: 25-OH vitamin D levels in the serum of the patients and healthy individuals were quantified with ELISA using the 25-OH vitamin D Total (Vit D-Direct) Test System ELISA Kit (Monobind Inc.®, United States, Product Code: 9425-300) on the EIA Reader Sirio S (Seac, Italy).

Techniques:

Journal: Journal of Medicine and Life

Article Title: 25-OH Vitamin D blood serum linkage with VDR gene polymorphism (rs2228570) in thyroid pathology patients in the West-Ukrainian population

doi: 10.25122/jml-2021-0101

Figure Lengend Snippet: rs2228570 genotypes as risk factors for reduced serum 25-OH vitamin D levels.

Article Snippet: 25-OH vitamin D levels in the serum of the patients and healthy individuals were quantified with ELISA using the 25-OH vitamin D Total (Vit D-Direct) Test System ELISA Kit (Monobind Inc.®, United States, Product Code: 9425-300) on the EIA Reader Sirio S (Seac, Italy).

Techniques:

Journal: International journal of cancer

Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

doi: 10.1002/ijc.30769

Figure Lengend Snippet: Downregulation of RNF20 mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.

Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

Techniques: Modification, Whisker Assay, Expressing, Western Blot, Transfection

Journal: International journal of cancer

Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

doi: 10.1002/ijc.30769

Figure Lengend Snippet: Changes of gene expression profile and gene set in A549, H1299, H460 cells upon downregulation of H2Bub1. (a) Unsupervised hierarchical clustering analysis of global expression profiles in lung epithelial cells upon RNF20 knockdown. The two top rows represent two independent siRNA repeats, RNF is for RNF20 siRNA, and C is for control siRNA. (b) qRT–PCR and (c) Western blot analysis for selected genes. The level of each gene in cancer cell transfected with RNF20 siRNA is the average ratio of triplicate samples, and is presented as the ratio to control sample transfected with scramble siRNA (* P < 0.05, compared with control). (d). Selected gene sets that were enriched upon RNF20 knockdown in these lung cancer cells.

Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: International journal of cancer

Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

doi: 10.1002/ijc.30769

Figure Lengend Snippet: Knockdown of RNF20 enhances proliferation, migration and inhibits retinoic acid-induced squamous differentiation of BEAS-2B cells. (a) Enriched gene sets in BEAS-2B cells with silenced RNF20 and decreased H2Bub1 (P < 0.05, FDR < 5%). (b) Western blot for RNF20 and H2Bub1, p53, Myc, and ALDH1A1 proteins. (c) Enhanced cellular proliferation and (d) migration upon RNF20 knockdown in BEAS-2B cells. After 72 h of siRNA transfection, cell proliferation was measured. At 48 h post-transfection, 5 x 104 cells transfected with either control or RNF20 siRNA were further subjected to transwell migration assay for 12 h, and the number of migrated cells was counted (*P < 0.05, compared to control siRNA). (e) qRT–PCR analysis of IVL, a squamous differentiation marker, mRNA in BEAS-2B cells transfected with control or RNF20 siRNA and cultured in the differentiating medium BEDM, data were presented as ratio to non-differentiating medium control (*P < 0.05, ** P < 0.01, compared with control). (f) Micrographs show morphology changes of BEAS-2B cells after transfection with control siRNA (left panel) or RNF20 siRNA (right panel) and then subcultured in BEDM. Arrows point to “fried egg” morphology of squamous cells in control siRNA-transfected BEAS-2B cells.

Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

Techniques: Migration, Western Blot, Transfection, Transwell Migration Assay, Quantitative RT-PCR, Marker, Cell Culture

Journal: International journal of cancer

Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

doi: 10.1002/ijc.30769

Figure Lengend Snippet: Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. (a) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. (b) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (*P < 0.05, compared to control siRNA). (c) Representative photomicrographs of the migration chamber at the times indicated in control or RNF20 siRNA-transfected cells is shown on the left, with quantitative analysis of migrated cells shown on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, compared with control siRNA). (d) Lower surface of Matrigel transwell membranes seeded with cancer cells previously transfected with indicated siRNAs, 24 h after incubation (left), and corresponding quantitative analysis of invading cells (right). Data are shown as mean values graphed for indicated cells on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, **P < 0.01, compared with control siRNA).

Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

Techniques: In Vitro, Migration, Western Blot, Transfection, Incubation

Journal: International journal of cancer

Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

doi: 10.1002/ijc.30769

Figure Lengend Snippet: Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in (a) A549 and (b). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. (c) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. (d) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P < 0.05; ** P < 0.01).

Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

Techniques: Western Blot, Flow Cytometry, Transfection, Labeling, Staining