Journal: Frontiers in Immunology

Article Title: MSC therapy ameliorates experimental gouty arthritis hinting an early COX-2 induction

doi: 10.3389/fimmu.2023.1193179

Figure Lengend Snippet: Ad-MSC modulates pro- and anti-inflammatory cytokine profile in synovial membranes of arthritic rabbits. Representative western blot of pro-inflammatory cytokines (A) COX-2, TNF, IL-6 and M2 anti-inflammatory cytokines levels (B) IL-10 and TGF-β. EZ blue staining was used as protein loading control and to normalize the results, which are expressed as a fold-change of the Control group. Bars show the mean and SEM. COX-2, Ciclooxygenase-2; IL, interleukin; TGF-β, tumor growth factor-β, TNF-α, tumor necrosis factor α; MSC, mesenchymal stem cells; MSU, monosodium urate.

Article Snippet: The following primary antibodies were applied overnight at 4°C: anti-human COX-2 (Santa Cruz Biotechnology, Dallas TX, USA), anti-rabbit IL-6, anti-rabbit TNF, anti-rabbit IL-10, anti-rabbit TGF-β (all from Cloud-Clone Corp; 1/250 dilution); anti-human NLRP3 (AdipoGen, Liestal, Switzerland; 1/1000 dilution); anti-human Caspase-1 (Thermo Fisher Scientific, IL, USA; 1/500 dilution), anti-rabbit IL-18 and IL-1β antibodies (Cloud-Clone Corp, Houston TX, USA; 1/250 dilution).

Techniques: Western Blot, Staining

Journal: Cancer Science

Article Title: Quantitative proteomic analysis of mitochondria from human ovarian cancer cells and their paclitaxel-resistant sublines

doi: 10.1111/cas.12710

Figure Lengend Snippet: (a) Electron microscopy image of mitochondria morphology (10 000×). The purity of the mitochondria was confirmed using electron microscopy, including intactness and contamination. (b) Western blot analyses of total cell proteins (Cell) and mitochondrial preparations isolated from paclitaxel-sensitive SKOV3 cells (Smt), paclitaxel-resistant SKOV3 cells (TSmt), paclitaxel-sensitive A2780 cells (Amt), and paclitaxel-resistant A2780 cells (TAmt) for COX4 (mitochondrial marker), lamin-B (nuclear marker), flotillin-1 (cell membrane marker) and β-actin (cytoskeletal protein). There was little contamination in the mitochondria-enriched fractions.

Article Snippet: Mouse anti-human flotillin-1 and rabbit anti-human β-actin antibodies were obtained from eBioscience (San Diego, CA, USA).

Techniques: Electron Microscopy, Western Blot, Isolation, Marker, Membrane

Journal: Cancers

Article Title: Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors

doi: 10.3390/cancers15010259

Figure Lengend Snippet: Impact of CNVs in oncogenic-associated genes on corresponding protein levels. CNV-protein links were evaluated by Western blots on tumor lysates from passages (P1, P2, and P3) of the OS PDXs (HT72, HT77, HT87, and HT96). Baseline levels of selected proteins of interest were: RAD21, MYC (c-MYC), p53, Cyclin D3, Cyclin E1, p16 INK4A , PTEN, RAC1, HSP90Aβ1, γH2AX, total H2AX, p-RB1 (S795), total RB1, BRD4, CDK4, and CDK6. Vinculin served as the loading control and was probed from lysates extracted in urea or RIPA buffers since proteins of interest were efficiently extracted in different buffers (see materials and methods). Human vertebral mesenchymal stem cells (HVMSCs) and human osteoblasts-femoral (HOB-F) were included as normal healthy control cells. This experiment was conducted one time. Copy numbers for genes containing CNVs are the top of each immunoblot.

Article Snippet: The following antibodies were diluted in either 5% non-fat dry milk or 5% BSA in TBS-T per manufacturer’s instruction and used for detection: rabbit anti-RAD21 (130 kDa, cat# 4321, Cell Signaling Technology, Boston, MA, USA); mouse anti-c-MYC [9E10] (67 kDa, cat# sc-40, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-p53 [D0-1] (53 kDa, cat# sc-126, Santa Cruz Biotechnology Inc.); mouse anti-Cyclin D3 (31 kDa, cat# 2936, Cell Signaling Technology, Boston, MA, USA); mouse anti-Cyclin E1 (48 kDa, cat# 4129, Cell Signaling Technology, Boston, MA, USA); rabbit anti-CDKN2A/p16 INK4A (17kDa, cat# ab108349, Abcam, Waltham, MA, USA); rabbit anti-PTEN (54 kDa, cat# 9559, Cell Signaling Technology, Boston, MA, USA); rabbit anti-RAC1 (21 kDa, cat# 4651, Cell Signaling Technology, Boston, MA, USA); rabbit anti-HSP90Aβ1 (96 kDa, cat# NBP2-68937, Novus Biologicals, Centennial, CO, USA); anti-phospho-H2AX serine 139 [γH2AX-Ser139] (15 kDa, cat# 2577, Cell Signaling Technology, Boston, MA, USA); rabbit anti-total H2A.X [D17A3] (15 kDa, cat# 7631, Cell Signaling Technology, Boston, MA, USA); rabbit anti-RB1 [D20] (110 kDa, cat# 9313, Cell Signaling Technology, Boston, MA, USA); rabbit anti-phospho-RB1 Ser795 (110 kDa, cat# 9301, Cell Signaling Technology, Boston, MA, USA); rabbit anti-BRD4 [E2A7X] (200 kDa, cat# 13440, Cell Signaling Technology, Boston, MA, USA); rabbit anti-CDK4 [D9G3E] (30 kDa, cat# 12790, Cell Signaling Technology, Boston, MA, USA); rabbit anti-CDK6 [D4S8S] (36 kDa, cat# 13331, Cell Signaling Technology, Boston, MA, USA); rabbit anti-vinculin [E1E9V] (124 kDa, cat# 13901, Cell Signaling Technology, Boston, MA, USA), and rabbit anti-GAPDH [14C10] (37 kDa, cat# 2118, Cell Signaling Technology, Boston, MA, USA).

Techniques: Western Blot, Control

Journal: Cancer Science

Article Title: Rigosertib induces cell death of a myelodysplastic syndrome-derived cell line by DNA damage-induced G2/M arrest

doi: 10.1111/cas.12605

Figure Lengend Snippet: Rigosertib causes aberrant multiplication of centrosomes and abnormal spindle assembly in MDS-L cells. (a) HL-60 and MDS-L cells were treated with indicated concentrations of rigosertib for 24 h, and protein lysates were analyzed by immunoblotting analysis for the detection of aurora A kinase, aurora B kinase and phospho-aurora A kinase with each antibody. The amount of alpha-tubulin is shown as a loading control. (b) Immunofluorescence staining of rigosertib-treated MDS-L cells. Cells were treated with or without 50 nM rigosertib for 24 h and immunostained by anti-aurora A kinase followed by AlexaFluor488(green) -conjugated secondary antibody and AlexaFluor555 (red)-conjugated rabbit polyclonal anti-β-tubulin antibody. The chromosome area was stained with DAPI (blue) (original magnification ×1000). Representative images of normal and abnormal mitotic patterns are shown. (c) The location of aurora A kinase in mitotic cells was classified into normal metaphase, normal anaphase and telophase, and deregulated mitotic pattern, and each rate (percentage) is indicated below from counting more than 500 cells at M phase in each concentration. Rigosertib 0 nM: normal metaphase 66%, normal anaphase and telophase 25%, and deregulated mitotic pattern 9%. Rigosertib 50 nM: normal metaphase 12%, normal anaphase and telophase 14%, and deregulated mitotic pattern 74%. Rigosertib 100 nM: normal metaphase 1%, normal anaphase and telophase 1%, and deregulated mitotic pattern 98%.

Article Snippet: Spindle assembly was detected with AlexaFluor 555-conjugated rabbit polyclonal anti-β-tubulin antibodies (Sigma-Aldrich).

Techniques: Western Blot, Immunofluorescence, Staining, Concentration Assay

Journal: Virus Research

Article Title: Cholesterol 25-hydroxylase suppresses porcine deltacoronavirus infection by inhibiting viral entry

doi: 10.1016/j.virusres.2021.198306

Figure Lengend Snippet: PDCoV infection upregulates CH25H expression in IPI-FX cells. (A, B) IPI-FX cells were infected with different doses of PDCoV (MOI 0.25, 0.5, and 1). Uninfected cells were used as a negative control. Samples were collected at 12 hpi and CH25H mRNA and protein levels were determined by qRT-PCR ( A ) and western blotting ( B ), respectively. ( C, D ) IPI-FX cells were inoculated with PDCoV (MOI 1) and samples were collected at 6, 12, and 18 hpi. CH25H mRNA and protein levels were assessed by qRT-PCR ( C ) and western blotting ( D ), respectively. PDCoV infection was verified by western blotting with an anti-PDCoV N protein antibody. β-actin used as a sample loading control. All data were presented as the means ± standard deviations of three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: An anti-rabbit-β-actin monoclonal antibody (ABclonal, Wuhan, China) was used to assess the expression of β-actin and to confirm equal loading of protein samples.

Techniques: Infection, Expressing, Negative Control, Quantitative RT-PCR, Western Blot, Control