Journal: Breast Cancer Research and Treatment

Article Title: Vitamin D and androgen receptor-targeted therapy for triple-negative breast cancer

doi: 10.1007/s10549-016-3807-y

Figure Lengend Snippet: Effect of AR and/or VDR therapy on cancer stem cell and differentiation phenotype: a the effect of DHT and calcitriol on tumorsphere formation in TNBC HR2-av cell lines MFM-223 and CAL-148. The cells were trypsinized and single cell suspensions were plated into six-well low adhesion plates in 4-ml sphere media (seeding: MFM-223 10,000 and CAL-148 6000) containing DHT (10 nM, black bars ) or calcitriol (MFM-223 25 nM and CAL-148 10 nM, white bars ), or DHT + calcitriol ( hatched bars ). After 2 weeks during which spheres are formed from single cells, the plates were stained with iodo-nitrotetrazolium chloride overnight and the spheres were counted using GelCount. Y -axis represents the TFE, tumorsphere formation efficiency (number of spheres formed per the number of cells seeded in a well) represented as fold change in comparison to control group (no treatment, vehicle only). The statistical significance of the drug treatments was determined using two-tailed Student’s T -test. Error bars represent mean ± SEM. * P < 0.05, ** P < 0.01. b AldeFluor assay on HR2-av cell lines MFM-223 and CAL-148 after AR and VDR treatment. The cells were treated as described in panel a for 8 days with media change containing fresh drug every 3 days and ALDH + population was examined by AldeFluor assay kit using FACS profiles representing ALDH + population before ( top ) and after ( bottom ) co-treatment. The graph shows ALDH + cells plotted as percentage in each treatment group. Gray bar vehicle treatment, black bar DHT, white bar Cal, hatched DHT and Cal combination. Error bars represent mean ± SEM. The statistical significance of the drug treatments was determined using two-tailed Student’s T -test. * P < 0.05, ** P < 0.01. c , d Western blot analysis of differentiation and cancer stem cell markers in HR2-av cell lines MFM-223 and CAL-148 after DHT and calcitriol treatment. The cells were treated with DHT (10 nM) and/or calcitriol (MFM-223: 25 nM and CAL-148: 10 nM) for 8 days with media including drug every 3 days. The cell lysates with 30 µg protein were loaded in each lane of a 4–15 % SDS-PAGE gel and after transfer to nitrocellulose membrane, it was probed with the following antibodies at appropriate dilutions: E-cadherin (BD Biosciences 61081), vimentin (Sigma 5255), cytokeratin 5 (Abcam ab75869), cytokeratin 18 (Thermofisher Scientific MS-142-P), Claudin-4 (Invitrogen 32-9400), ALDH1A1 (Cell Signalling mAb12035), CD326 (Abcam ab32392), CD49f (ABD Serotec MCA1457), CD133 (Sigma C9493), CD24 (Santa Cruz SC-53660), CD166 (Abcam ab49496), Nanog (Abcam ab109250), and Musashi (Abcam ab52865). β-Actin or β-tubulin were used as loading controls. e mRNA expression of stem cell markers. MFM-223 cells were treated with 10 nM DHT and/or 25 nM calcitriol for 96 h. The mRNA was quantified using a PCR-based array (Qiagen). The expression level of each gene was determined by comparing the treatment to vehicle control, and genes with more than 2-fold change are presented here in a Heatmap generated using conditional formatting in Microsoft Excel. Scale bar red represents high expression, green represents low expression

Article Snippet: The cell lysates with 30 μg protein were loaded in each lane of a 4–15 % SDS-PAGE gel and after transfer to nitrocellulose membrane, it was probed with the following antibodies at appropriate dilutions: E-cadherin (BD Biosciences 61081), vimentin (Sigma 5255), cytokeratin 5 (Abcam ab75869), cytokeratin 18 (Thermofisher Scientific MS-142-P), Claudin-4 (Invitrogen 32-9400), ALDH1A1 (Cell Signalling mAb12035), CD326 (Abcam ab32392), CD49f (ABD Serotec MCA1457), CD133 (Sigma C9493), CD24 (Santa Cruz SC-53660), CD166 (Abcam ab49496), Nanog (Abcam ab109250), and Musashi (Abcam ab52865). β-Actin or β-tubulin were used as loading controls. e mRNA expression of stem cell markers.

Techniques: Staining, Comparison, Control, Two Tailed Test, Western Blot, SDS Page, Membrane, Expressing, Generated

Journal: Open life sciences

Article Title: Effects of phytoestrogens combined with cold stress on sperm parameters and testicular proteomics in rats.

doi: 10.1515/biol-2022-0531

Figure Lengend Snippet: Figure 3: ITGA6 was downregulated, Col6α1 and Col6α2 were upregulated in the testis of the model group exposed to an estrogen-enriched diet and cold for 24 weeks. (a) Immunofluorescence staining for ITGA6 (red, 200×), Col6α1 (green, 100×), Col6α2 (red, 100×); nuclei were stained with DAPI (blue). (b, c, and d) Representative western blot results for ITGA6, Col6α1, Col6α2. (e, f, and g) Relative expression of ITGA6, Col6α1, Col6α2 compared with GAPDH. Protein lysates were obtained from the testis of either model (M) or control rat group (N). Protein expression of ITGA6, Col6α1, and Col6α2 was evaluated by western blotting using specific antibodies for the target proteins, and the signal was then quantified using Image J. (h, i, and j) qRT-PCR of ITGA6, Col6α1, and Col6α2 in the testis of rats in the two groups. Values are expressed as the mean ± SD. Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to the control group.

Article Snippet: After blocking in 5% nonfat milk for 2 h at 37°C, the membrane was incubated with the primary antibody overnight at 4°C: integrin alpha 6 (ITGA6) (1:1,000; Boster, BA3356, Wuhan, China), alpha 1 chains of type VI collagen (Col6α1) (1:1,000; Boster, BM4748, Wuhan, China), alpha 2 chains of type VI collagen (Col6α2) (1:500; Affinity, DF3552, Zhenjiang, China), then with a Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG(H + L) secondary antibody (1:5,000; ZSGB-BIO, ZB2301, Beijing, China) for 2 h at 37°C.

Techniques: Staining, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Cell reports

Article Title: 2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity

doi: 10.1016/j.celrep.2021.110220

Figure Lengend Snippet:

Article Snippet: Rat monoclonal anti-CD49F (Clone GoH3) , Fluidigm , Cat# 3164006B.

Techniques: Recombinant, Control, Blocking Assay, Suspension, Electron Microscopy, Purification, Whole Genome Amplification, SYBR Green Assay, Labeling, Staining, Methylation Sequencing, Methylated DNA Immunoprecipitation, Biomarker Discovery, Software, Pyromark Assay

Journal: Cancer research

Article Title: RAD51AP1 deficiency reduces tumor growth by targeting stem cell self-renewal

doi: 10.1158/0008-5472.CAN-19-3713

Figure Lengend Snippet: RAD51AP1 KD potentiates chemotherapy and radiation therapy response by inhibiting stem cell self-renewal signaling. A, Percent apoptosis (Annexin V+/PI−) was calculated in CAL51-pLKO.1 and CAL51-RAD51AP1shRNA transfected cells with PTX (10 nM) and IR (10 Gy), respectively. B and C, Similarly, percent apoptosis was calculated in AT-3-pLKO.1 and AT-3-RAD51AP1shRNA transfected cells as well as cell isolated from the mammary tumor tissues obtained from Rad51ap1+/+-C3(1)-Tg and Rad51ap1+/+-C3(1)-Tg mice with PTX (10 nM) and IR (10 Gy), respectively. Values are expressed as Mean ± SD of three independent experiments or three mice in each with triplicated values = 9 datasets. D, RAD51AP1 mRNA and RAD51AP1 protein expression in PTX-S (sensitive) and PTX-R (resistant) CAL51 cell lines as measured by qPCR and western blot, respectively. n=4 independent clones. E, Relative RAD51AP1 transcript expression in CAL51-PTX-R cells transfected with pLKO.1 and RAD51AP1shRNA. F, percent apoptosis was calculated in PTX-R-pLKO.1- and PTX-R-RAD51AP1shRNA-transfected cells treated with and without PTX (10 nM for 48 h). G and H, Representative FACS contour plots and quantification of BCSC (CD49f+CD24+) in CAL51-PTX-R-pLKO.1 and CAL51-PTX-R-RAD51AP1shRNA cells. I, Relative expression of stem cell self-renewal genes in CAL51-PTX-R-pLKO.1 and CAL51-PTX-R-RAD51AP1shRNA cells.

Article Snippet: We used the following antibodies for this study: CD49f and CD24 (STEMCELL Technology and eBioscience), CD45, CD31, CD49f, CD44 (BD bioscience), and Ter119 and CD61 (eBioscience).

Techniques: Transfection, Isolation, Expressing, Western Blot, Clone Assay

Journal: Cancer research

Article Title: RAD51AP1 deficiency reduces tumor growth by targeting stem cell self-renewal

doi: 10.1158/0008-5472.CAN-19-3713

Figure Lengend Snippet: RAD51AP1 KD reduces cancer stem cell self-renewal signaling. A, Heat map generated from the qPCR dataset for epithelial differentiation, breast cancer stem cell marker, pluripotency, and EMT/Metastasis of CAL51-pLKO.1 and CAL51-RAD51AP1(#2) cells (n=3 independent clones with triplicates = 9 dataset in each). B, Relative expression of breast cancer stem cell self-renewal genes in AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#3) by qPCR analysis. (n=3 independent clones with triplicates = 9 dataset in each). C, Relative expression of breast cancer stem cell self-renewal genes in Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg by qPCR analysis. (n=3 mice with triplicates =9 dataset in each). D and E, Representative FACS dot plots and quantification of BCSC (CD49f+CD24+) in CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) cells. F and G, Representative FACS dot plots and quantification of BCSC in Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice. H and I, Representative images of tumorospheres generated form Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice and quantification of primary, secondary, and tertiary tumorospheres number (n=3–5 mice in each).

Article Snippet: We used the following antibodies for this study: CD49f and CD24 (STEMCELL Technology and eBioscience), CD45, CD31, CD49f, CD44 (BD bioscience), and Ter119 and CD61 (eBioscience).

Techniques: Generated, Marker, Clone Assay, Expressing