Journal: PLoS ONE

Article Title: Identification of the best housekeeping gene for RT-qPCR analysis of human pancreatic organoids

doi: 10.1371/journal.pone.0260902

Figure Lengend Snippet: The relative transcriptional level of the two ductal markers EpCAM and SOX9 in each sample condition considered was normalized with the best and the worst reference gene (RPL13A and ACTB, respectively), the geometric mean (Geo.mean) of two selected HKGs by NormFinder (PPIA and UBC) and one commonly used gene (GAPDH). One-way analysis of variance followed by Dunnett’s post-hoc test for multiple comparisons was used.

Article Snippet: We stained the samples using 1:200 FITC mouse Anti-Human Epithelial Cell Adhesion Molecule (EpCAM; BD, cat. #347197), 1:100 Alexa Fluor® 647 Mouse Anti- SRY-box transcription factor 9 (Sox9; BD, cat. #565493) and 1:2000 FITC-conjugated lectin from Ulex europaeus (UEA1) (Sigma-Aldrich, cat. #L9006).

Techniques:

Journal: bioRxiv

Article Title: Glycogen synthase kinase 3 alpha/beta deletion induces precocious growth plate remodeling and cell loss in mice

doi: 10.1101/2020.04.04.025700

Figure Lengend Snippet: Presence of Sox9 positive cells in Gsk3a/Gsk3b KO and control mice at P36, P43 and P58 was assessed by immunohistochemistry on frontal knee joint sections (A). Bone marrow cavity-like regions are highlighted in black dotted line. Localized expression and loss of GSK3alpha (B&C) and GSK3beta (D&E) in Gsk3a/Gsk3b KO mice was evaluated by IHC at P36 (B&D) and P43 (C&E). Images are representative of 4-7 mice per group for Sox9, and 3-4 mice per group were used for GSK3alpha and GSK3beta at both time points.

Article Snippet: Primary antibodies used for immunolabelling are as follows: goat polyclonal Sox9 (AF-3075, R&D Systems), rabbit monoclonal GSK3alpha (ab40870, Abcam), rabbit polyclonal GSK3beta (12456S, Cell Signaling Technologies), mouse monoclonal type II collagen (Col2; 10R-C135b, Fitzgerald), rabbit polyclonal osteocalcin (ab93876, Abcam), rabbit polyclonal beta-catenin (bs-1165R, Bioss Antibodies) and rabbit polyclonal GLI1 (PA5-72942, Invitrogen, Thermo Fisher).

Techniques: Control, Immunohistochemistry, Expressing

Journal: Oncotarget

Article Title: Blocking the formation of radiation–induced breast cancer stem cells

doi:

Figure Lengend Snippet: Non-transfected BC, BC cells transfected with NF-κB p65 siRNA or control siRNA were fraction-irradiated (3.75 Gy/day × 5 days). Non-transfected, fraction-irradiated BC cells were cultured for 24 hours, followed by treatment with different agents as indicated for an additional 24 hours. siRNA transfected fraction-irradiated BC cells were cultured for 24 hours, followed by treatment with NAC for an additional 24 hours. The expression of ERBB2, SOX9 and MYC (genes named by HUGO gene database) was analyzed by real-time quantitative (q) RT-PCR. Gene expression values obtained from treated cells were compared with those obtained from untreated cells, which were standardized to a value of 1. The results of the comparison were expressed in terms of fold change ± SD and P values are shown (A). Treated cells were lysed and analyzed by Western blotting for expression of HER2/Neu, SOX9 and c-myc (proteins named by NCBI protein database) (B). Mammosphere formation assays were performed with cells treated as indicated. Pictures were taken on day 10 (SUM149) and on day 14 (MDA-MB-231). *** indicates P<0.001(C). The concentrations of NF-κBi and ROSi used were1 μM and 10 mM, respectively. All of the experiments were performed in duplicate and repeated twice, and the means ± SD are shown.

Article Snippet: Antibodies for IHC staining: mouse Neu-specific rabbit Ab (#sc-284) (1:250 dilution) and mouse SOX9-specific rabbit Ab (#sc-20095) (1:200 dilution) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Irradiation, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Comparison, Western Blot

Journal: Oncotarget

Article Title: Blocking the formation of radiation–induced breast cancer stem cells

doi:

Figure Lengend Snippet: Mouse 4T1 cells were injected subcutaneously (s.c.) into the right hind leg of 28 mice. On day 7, when the tumors reached 5 mm in diameter, mice were assigned in a stratified randomized manner to 4 groups (n=7 per group) and the treatments were initiated. Group 1 (Vehicle control) was treated orally with the vehicle control olive oil. Group 2 (IR) was administered a single dose of IR (20Gy) on day 10. Group 3 (DSF) was treated orally with DSF once daily for 8 days. Group 4 (IR+DSF) was treated orally with DSF, once daily for 8 days and mice were administered a single dose of IR on day 10. Tumor sizes were monitored twice per week. Mean tumor volumes of each group ± SD and P values for comparison between groups are shown (A). On day 15, two mice from each group were sacrificed and their tumors (Day 15 tumors) were collected for analysis of intratumoral levels of stemness gene expression. Total RNA was extracted from fresh 4T1 tumors and analyzed for mRNA of stemness genes by quantitative real-time qRT-PCR. Gene expression values obtained from treated tumors were compared with those obtained from untreated tumors, which were standardized to a value of 1. The results of the comparison were expressed in terms of fold change. The experiments were performed in duplicate and the means of fold change ± SD and P values are shown (B). The remaining 5 mice from each group were sacrificed on day 29 and tumors (Day 29 tumors) were collected for analysis of stemness genes using the same method as described above. The experiments were performed in duplicate and the means of fold change ± SD and P values are shown (C). Tumors from all mice collected on both days 15 and 29 were immunohistochemically (IHC) stained for protein products of stemness genes. Staining results of Neu, SOX9, c-myc and WNT3 were evaluated and expressed as positive cells in 10 high-power (200x) fields.P values for comparison between groups are shown (D). Day 15 tumors (n=2 per group) were digested to test intratumoral BCSCs/iBCSCs using mammosphere formation assays. The experiments were performed in duplicate and the means ± SD are shown. *** indicates P<0.001 (E). Day 29 tumors (n=5 per group) were digested for analysis of intratumoral BCSCs/iBCSCs using the same method as described above. The experiments were performed in duplicate and the means ± SD are shown. *** indicates P<0.001 (F).

Article Snippet: Antibodies for IHC staining: mouse Neu-specific rabbit Ab (#sc-284) (1:250 dilution) and mouse SOX9-specific rabbit Ab (#sc-20095) (1:200 dilution) were purchased from Santa Cruz Biotechnology.

Techniques: Injection, Control, Comparison, Gene Expression, Quantitative RT-PCR, Staining

Journal: BMC developmental biology

Article Title: Sonic hedgehog promotes chondrogenesis of rabbit bone marrow stem cells in a rotary cell culture system.

doi: 10.1186/s12861-019-0198-4

Figure Lengend Snippet: Fig. 5 The effect of Shh on cartilage related markers during differentiation induction in the RCCS environment. a-h RT-PCR analysis of Sox9, ACAN, collagen II, collagen X, RUNX2, ALP, PPAR-γ and leptin on days 7,14 and 21 during differentiation induction. i, j Expression of Sox 9, collagen II, ACAN, collagen X, RUNX2, ALP and PPAR-γ in the RCCS dimensional environment on days 10 and 21 of chondrogenesis. The representative results were from three independent experiments. Significant differences between the control group are indicated by * p < 0.05, **p < 0.01; differences between Shh and TGF-β transfection groups are indicated by # p < 0.05 or ## p < 0.01

Article Snippet: Antibodies recognising Ptc (Aviva Systems Biology, San Diego, CA, USA; 1:500), Smo (Aviva Systems Biology; 1:1,600), Gli1 (Biorbyt, Cambridgeshire, UK; 1:1,000), Sox9 (OriGene, Rockville, MD, USA; 1:500), collagen II (Novus Biologicals, Littleton, CO, USA; 1:200), ACAN (Novus Biologicals; 1:100), collagen X (Abcam; 1:500), RUNX2 (Abcam; 1:500), ALP (Abcam; 1:500), PPAR-γ (Santa Cruz Biotechnology; 1:1000) and GAPDH (Novus Biologicals; 1:2000) were used as primary antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Transfection