Journal: Molecular Pharmacology

Article Title: A Novel Gemini Vitamin D Analog Represses the Expression of a Stem Cell Marker CD44 in Breast Cancer

doi: 10.1124/mol.110.068403

Figure Lengend Snippet: Effects of BXL0124 on CD44 protein expression level in MCF10DCIS.com xenograft tumors in vivo. A, MCF10DCIS.com xenografted nu/nu mice were treated with DMSO or BXL0124 (0.1 μg/kg body weight) orally, and mammary tumors were collected at necropsy. Mammary tumors (n = 5) were pooled into either the control group or BXL0124-treated group for Western blot analysis against CD44, CD44v3, CD44v6, PCNA, and β-actin. B, a representative H&E staining in mammary tumors from MCF10DCIS.com xenografted nu/nu mice is shown (original magnification, 400×). C, a representative immunostaining against CD44 and PCNA in mammary tumors from MCF10DCIS.com xenografted nu/nu mice is shown (original magnification, 400×). Three mammary tumors from each group were selected and three representative areas from each tumor were analyzed for the expression of CD44 and PCNA. The mammary tumors immunostained against CD44 and PCNA were scored by four different levels of staining intensity and quantified by using Aperio Scan Scope. The data are presented as the mean ± S.D. (statistical analysis: *, p < 0.05; **, p < 0.01).

Article Snippet: The slides were incubated overnight at −4°C with CD44 primary antibody (1:50; Santa Cruz Biotechnology) or proliferating cell nuclear antigen (PCNA) primary antibody (1:1000; BD Pharmingen, San Diego, CA).

Techniques: Expressing, In Vivo, Western Blot, Staining, Immunostaining

Journal: Molecular Pharmacology

Article Title: A Novel Gemini Vitamin D Analog Represses the Expression of a Stem Cell Marker CD44 in Breast Cancer

doi: 10.1124/mol.110.068403

Figure Lengend Snippet: Effects of BXL0124 on CD44 protein expression level in MCF10DCIS.com breast cancer cells in vitro. A, MCF10DCIS.com cells were treated with increasing doses of 1α,25(OH)2D3 or BXL0124 (0.01, 0.1, 1.0, and 10 nM) for 24 h and analyzed for CD44 and PCNA protein expression levels by Western blot analysis. MCF10DCIS.com and MCF10CA1a cells were treated with BXL0124 (10 nM) for 24 h and analyzed for CD44 and PCNA protein expression levels by Western blot analysis. All splicing isoforms of CD44 were recognized by a CD44 antibody, which recognizes both CD44 standard and variants. B, MCF10DCIS.com cells were treated with DMSO or BXL0124 (10 nM) for 24 h and analyzed for CD44 expression level by confocal microscopy. C, MCF10DCIS.com cells were treated with DMSO control or BXL0124 (10 nM) for 24 h. The percentage of cells, which were categorized by the combination of CD44 and CD24 expression, was determined by flow cytometry. The experiment was repeated three times, and the data are presented as the mean ± S.D. D, MCF10DCIS.com cells were incubated without siRNA or with negative control siRNA or 1 μM concentration of each of two VDR siRNAs targeting different sequences in the VDR gene in Accell siRNA delivery medium for 72 h and followed by treatment with DMSO or BXL0124 (10 nM) for 24 h. The levels of CD44 and VDR protein were determined by Western blot analysis.

Article Snippet: The slides were incubated overnight at −4°C with CD44 primary antibody (1:50; Santa Cruz Biotechnology) or proliferating cell nuclear antigen (PCNA) primary antibody (1:1000; BD Pharmingen, San Diego, CA).

Techniques: Expressing, In Vitro, Western Blot, Confocal Microscopy, Flow Cytometry, Incubation, Negative Control, Concentration Assay

Journal: Antioxidants

Article Title: Oxidative Stress, NF-κB-Mediated Inflammation and Apoptosis in the Testes of Streptozotocin–Induced Diabetic Rats: Combined Protective Effects of Malaysian Propolis and Metformin

doi: 10.3390/antiox8100465

Figure Lengend Snippet: Expression of NF-κB (p65) protein ( a , b ) and mRNA ( c ); and the expression of TNF-α protein ( d , e ) and mRNA ( f ) in the testes of ( a ) NC, ( b ) DC, ( c ) D+MP, ( d ) D+Met and ( e ) D+MP+Met groups. For each biological replicate, a negative control was included, in which phosphate buffered saline was substituted for the primary antibody. A representative photomicrograph of the negative control is shown as ( f ) (magnification = ×100, scale bar = 200 µm). For quantitative data, values are mean ± SD, n = 8 (for staining intensity) and n = 6 (for mRNA expression). a p < 0.05 versus NC, b p < 0.05 versus DC, c p < 0.05 versus D+MP, d p < 0.05 versus D+Met (one-way ANOVA followed by Tukey’s post-hoc test). NC: normoglycaemic control, DC: diabetic control, D+MP: diabetic rats treated with Malaysian propolis, D+Met: diabetic rats treated with metformin, D+MP+Met: diabetic rats treated with Malaysian propolis + metformin, NF-κB: nuclear factor kappa B, TNF-α: tumour necrosis factor alpha.

Article Snippet: Testes sections were incubated with polyclonal primary antibodies for NF-kB(p65) (1:80), IL-1β (1:80), IL-10 (1:40), caspase-3 (1:150) and PCNA (1:50) (Cloud-Clone Corp, Houston, TX, USA) overnight at 4 °C, while incubation with TNF-α (1:120) polyclonal primary antibody was performed for 1 h at room temperature.

Techniques: Expressing, Negative Control, Staining

Journal: Translational Respiratory Medicine

Article Title: A linear polyethylenimine mediated siRNA-based therapy targeting human epidermal growth factor receptor in SPC-A1 xenograft mice

doi: 10.1186/2213-0802-1-2

Figure Lengend Snippet: After repeated i.p. administration, tumor PCNA protein expression detected by IHC. (A) and apoptosis examined by TUNEL assay (B) . Quantitative analysis data revealed 37% reduce in cell proliferation index in LPEI/siRNA-EGFR group compared with other two groups (C) , and more than twofold increase in apoptosis index under LPEI /siRNA-EGFR treatment (D) . All the results are shown as the mean±standard error of numbers obtained from 6 animals in each group. *P < 0.05.

Article Snippet: After protein denature, using microwave and non-specific biding blocking with normal goat serum for 20 min at RT, sections were incubated with primary antibodies against EGFR or PCNA (both diluted to 1:100, Maixin-Bio, China) overnight at 4°C.

Techniques: Expressing, TUNEL Assay