Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: p53 expression is regulated in a dose dependent manner by Dox . (A and B) Cells treated with Dox for 48 h were harvested and western blot for p53 was performed. (C) No induction of p53 in response to Dox was observed in HTet43GFP cells. (D) Fold induction of p53 was calculated by densitometric analysis of p53 western blot in HTet23p53, HTet26p53 and HTet43GFP cells taking control = 1with normalization to β-Actin or β -Tubulin.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Control

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: p53 expression is regulated in a time dependent manner . (A, B and C) HTet23p53, HTet26p53 and HTet43GFP cells were treated with 1000 ng/ml Dox for indicated time points and western blots were performed for p53. (D) Fold induction was calculated by densitometric analysis of p53 in HTet23p53, HTet26p53 and HTet43GFP cells taking 0 h = 1 with normalization to β -Tubulin or GAPDH.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: p53 over expression did not reduce colony number . Five thousand cells were plated in low-melting agarose containing complete medium and allowed to grow for 30 days. (A, B and C) Cells were stained with crystal violet and photographs were taken under microscope for HTet23p53, HTet26p53 and HTet43GFP plates. (D) Colonies were counted and average number of colonies was plotted vs Dox concentration. Bar represents average of colonies from two representative fields (± SE).

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Over Expression, Staining, Microscopy, Concentration Assay

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is stable . (A and B) p53 was overexpressed with 1000 ng/ml of Dox for 48 h or not overexpressed and western blotswere performed after indicated time of Chx treatment in HTet23p53 and HTet26p53 cells. (C) Western blot for p53 was performed with or without addition of 1000 ng/ml Dox for 48 h followed by Chx treatment for indicated time points in HTet43GFP cells.(D) Graphical representation of percentage of p53 protein remaining after indicated time points in HTet23p53 and HTet26p53 cells by densitometric analysis following normalization with β-Actin. Protein percentage for 0 h Chx was taken as 100.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is not stabilized by inhibition of proteasomal degradation . Cells were treated with proteasomal inhibitors (MG132 or Lactacystin) 1 h prior to Dox addition and incubated for 48 h. Thereafter, cells were treated with Chx for indicated time points and western blots were performed. (A) MG132 treatment in HTet23p53 and HTet26p53 cells and p53 protein expression. (B) Lactacystin treatment in HTet23p53 and HTet26p53 cells and p53 protein expression.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Incubation, Western Blot, Expressing

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is functionally impaired by HPV E6 . (A) HTet23p53, HTet26p53 or HTet43GFP cells were transfected with vector or HPV18 E6 plasmid and 18 h post transfection cells were treated with OA 1 h prior to Dox addition. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) HTet26p53 cells were transfected with HPV18 E6 plasmid and treated as mentioned in A and immunoprecipitation was performed first with E6 antibody (first IP) and secondly by p53 antibody. p53 or E6 was detected in immunoprecipitated complex. (C) p53 post-IP (second IP) following E6 IP was performed and p53 or phospho-p53detection by western blotting was performed. (D) Cells were transfected with p21luciferase construct with or without HPV18 E6 plasmid and treated as mentioned in A. Luciferase assay was performed and luciferase/GFP reading was plotted. Bar represents results from an experiment done in triplicate. (± SE). * Indicates P < 0.05.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, MTT Assay, Immunoprecipitation, Western Blot, Construct, Luciferase

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is active and is made non-functional by HPV 18 E6 in H1299, a p53 and E6 null cell line . (A) H1299 cells were transfected with pC53-SN3 and/or HPV18 E6 plasmids and 18 h post transfection cells were treated with OA. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) H1299 cells were transfected with pC53-SN3 and/or HPV18E6 plasmids and 18 h post transfection cells were treated with OA for 48 h and western blot was performed for pSer46p53 and p53.(C) Densitometric analysis was performed by normalization with p53 and ratio of pSer53 and p53 was plotted.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Functional Assay, Transfection, MTT Assay, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: cAMP inhibits the IR-induced p53 accumulation in an Epac-independent fashion. (A) Reh cells were treated with forskolin or 8CPT-cAMP for 30 minutes before exposure to 10 Gy of IR. Cells were harvested at the indicated times after IR, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. p53 bands were subsequently quantified by densitometric analysis, and fold increase of p53 band intensity was calculated relative to the unirradiated control. One representative experiment of three is shown. (B) Human peripheral CD4+ T cells, U2OS and MCF-7 were treated with forskolin or 8-CPT-cAMP for 30 minutes before exposure to IR. After 4 hours, cells were harvested, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. One representative experiment of three is shown. (C) Reh cells were treated with the indicated concentrations of 8-CPT-cAMP or 8-pCPT-2′-O-Me-cAMP for 30 minutes before exposure to IR. After 4 hours, the cells were harvested, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: p53 mRNA steady-state levels are not affected by IR or cAMP. Reh cells were treated with forskolin 30minutes before 10 Gy IR. Cells were harvested at the indicated times after IR, RNA was isolated and subjected to Northern blot analysis with a p53 cDNA probe. RNA isolated formHCT116 p53+/+ and p53-/- cells was used as control for the specificity of the cDNA probe.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Isolation, Northern Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: cAMP inhibits the IR-induced stabilization of the p53 protein. Reh cells were treated with forskolin (60 µM) or 8-CPT-cAMP (200 µM) for 30 minutes before IR. Four hours after IR, cells were treated with cycloheximide (CHX; 25 µg/ml) and then harvested at the indicated times. Whole-cell lysates were prepared and analyzed by immunoblot analysis with DO-1 and antiactin antibodies. Upper panel shows one representative experiment of four. Lower panel: the immunoblots represented in the upper panel were scanned, and the intensity of the p53 protein bands was quantitated and plotted with the value obtained for cells not treated with CHX set as 1. Values were normalized with those of actin (n = 4).

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: cAMP inhibits p53 accumulation in a proteasome-dependent manner and counteracts IR-induced reduction of p53 ubiquitination. (A) Reh cells were preincubated with (upper panel) or without (lower panel) MG-132 for 2 hours before treatment with forskolin or 8-CPT-cAMP for 30 minutes. Cells were then exposed to IR, harvested at the indicated times post-IR, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. Vertical lines have been inserted to indicate repositioned gel lanes. Lower panel is shown as comparative data on the effect of MG-132 on p53 levels. (B) Upper panel: Reh cells were pretreated with MG-132 for 2 hours before addition of forskolin. After 30 minutes, cells were exposed to IR, harvested after 4 hours, and then subjected to immunoblot analysis with DO-1 and antiactin antibodies. Lower panel: Reh cells were treated as described for the upper panel. Whole-cell extracts were prepared and immunoprecipitated (IP) with FL-393 antibody. The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblot analysis with antibodies against ubiquitin. Subsequently, the blot was stripped and then reprobed with DO-1.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot, Immunoprecipitation, SDS Page

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: The inhibitory effect of cAMP on p53 accumulation requires functional HDM2 and involves inhibition of IR-induced dissociation of the p53-HDM2 complex. (A) Reh cells were treated with Nutlin-3a for 10 minutes before the addition of 8-CPT-cAMP, forskolin, or corresponding volumes of their solvents dH2O or DMSO, respectively. After 30 minutes, cells were exposed to IR and incubated for an additional 4 hours. Whole-cell lysates were then prepared and analyzed by immunoblot analysis with the DO-1 and antiactin antibodies. The immunoblot shows one representative experiment of three. The histogram depicts the average densitometric value of the p53 protein bands. Values from cAMP-treated samples have been normalized to their relevant solvent controls, whose values were set to 100% (n = 3). (B) Reh cells were transfected with control siRNA or siRNA against HDM2. After 24 hours, cells were cultured in the presence or absence of forskolin for 30 minutes before exposure to IR and incubated for an additional 4 hours. Whole-cell lysates were then prepared and analyzed by immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2), DO-1, and antiactin antibodies. (C) Reh cells were treated with forskolin for 30 minutes before exposure to IR. Cells were then harvested at the indicated times after IR and subjected to immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2) and antiactin antibodies. The immunoblot shows one representative experiment of seven. The immunoblots represented above were scanned, and the intensity of the p53 protein bands was quantitated and plotted with the value obtained for untreated cells set as 1 (n = 7). (D) Reh cells were treated as in C and analyzed by immunoblot analysis with the indicated antibodies. One representative experiment of three is shown. (E) Reh cells were pretreated with MG-132 for 2 hours before addition of forskolin. After 30 minutes of forskolin treatment, cells were exposed to IR and then harvested at the indicated times. Whole-cell extracts were prepared and immunoprecipitated with FL-393. The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblot analysis with the anti-HDM2 (a mixture of SMP14, IF2, and 4B2) and DO-1 antibodies. The immunoblot shows one representative experiment of seven. The intensity of protein bands was quantified densitometrically, and the ratio of signal intensity for HDM2 relative to p53 was calculated and plotted with the value obtained for untreated cells set as 1 (n = 7; at all three time points, there is a significant difference between cells treated with IR alone and those treated with IR + forskolin, P < .05). Panel marked input represents Western blots for HDM2, p53, and actin in cell extracts before immunoprecipitation. Vertical lines have been inserted to indicate repositioned gel lanes.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Functional Assay, Inhibition, Incubation, Western Blot, Transfection, Cell Culture, Immunoprecipitation, SDS Page

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: Effects of cAMP on p53 phosphorylation. Upper panel: Reh cells were treated with MG-132 (20 µM) for 2 hours before addition of forskolin (60 µM). After 30 minutes, cells were exposed to 10 Gy of IR and harvested at the indicated times. For the examination of p53 phosphorylation at S15 and S20, whole-cell lysates were subjected to immunoblot analysis with phospho-specific antibodies against p53 phosphorylated at S15 or S20. Subsequently, the membranes were stripped and reprobed with DO-1 to detect total p53 protein. One representative experiment of five is shown. For detection of p53 phosphorylated at T18, whole-cell lysates were immunoprecipitated with DO-1, and the recovered proteins were immunoblotted with phospho-specific antibodies against p53 phosphorylated at T18. The blot was then stripped and reprobed with total p53 (FL-393) antibody. One representative experiment of four is shown. Lower panel: The immunoblots represented in the upper panel were scanned, and the intensity of protein bands was quantified densitometrically. The ratio of signal intensity for phosphorylated p53 at S15, S20, or T18 relative to total p53 was then calculated, and the obtained values were normalized to the value obtained for cells at time 0 (S15 and S20, n = 5; T18, n = 4).

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: Inhibition of p53-induced cell death requires binding of HDM2 to p53. Reh cells were treated with forskolin for 30 minutes before addition of Nutlin-3a or exposure to 10 Gy IR. After 4 hours, a portion of cells were harvested and subjected to immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2), DO-1, and antiactin antibodies. The immunoblot shows one representative experiment of four. The remaining cells were analyzed for cell death by PI staining at 20 hours after IR (n = 4, *P < .01, relative to cells treated with IR only).

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Inhibition, Binding Assay, Western Blot, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: Model depicting how cAMP regulates the DNA damage-induced accumulation of p53.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques:

Journal: Oncology Letters

Article Title: Comparison of the expression of TGF-β1, E-cadherin, N-cadherin, TP53, RB1CC1 and HIF-1α in oral squamous cell carcinoma and lymph node metastases of humans and mice

doi: 10.3892/ol.2017.7456

Figure Lengend Snippet: Expression of tumor protein (TP)53 at different stages in the progression of oral carcinoma in human and mouse samples. (A) Photomicrographs of immunohistochemical positive expression of TP53 (scoring 2/3) in normal and dysplastic oral mucosa, OSCC and submandibular lymph node in lymphatic nodes metastatic carcinoma (magnification, ×200). (B and C) Data relative to the expression of TP53 at the different cancer stages. (D) Positive expression rate of TP53 at different cancer stages in human and mouse samples. *Compared with normal stage in human samples, P<0.05; ∆compared with normal or dysplasia stages in human samples, P<0.05; #compared with normal or dysplasia stages in mouse samples, P<0.05.

Article Snippet: The antibodies used in the present study were as follows: Mouse monoclonal antibodies for E-cadherin and p53 (ZSGB-Bio, China), rabbit polyclonal antibodies for TGF-β1 and HIF-1α (Boster Biological Technology, China), mouse monoclonal antibody for N-cadherin (Santa Cruz, USA), and rabbit polyclonal antibodies for RB1CC1 (Proteintech Group, China).

Techniques: Expressing, Immunohistochemical staining

Journal: Respiratory Research

Article Title: HGF alleviates septic endothelial injury by inhibiting pyroptosis via the mTOR signalling pathway

doi: 10.1186/s12931-020-01480-3

Figure Lengend Snippet: HGF alleviated endothelial pyroptosis in vitro. EA.hy926 cells were stimulated with LPS (2.5 μg/mL) with or without Lipo2000 (2 μL/mL), followed by administration of HGF (25 ng/mL) immediately and 6 h later. a Bright-field image of treated EA.hy926 cells from randomly selected fields of view, in which morphological changes characteristic of pyroptosis were assessed; scale bar = 100 μm; b Relative paracellular permeability of single-layer EA.hy926 cells exposed to different treatments and analysed by FITC-dextran; c IL-1β in the supernatant of treated EA.hy926 cells was measured by ELISA; d LDH release assay was performed to measure pyroptosis of the treated EA.hy926 cells, n = 3, * P < 0.05, ** P < 0.01; e CASP-1, CASP-1-P10, GSDMD and GSDMD-N in the treated EA.hy926 cell homogenates were measured by Western blot; n = 3

Article Snippet: The mouse monoclonal antibodies against gasdermin D (GSDMD) (sc-393,581) and β-actin (sc-517,582) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vitro, Permeability, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot

Journal: Respiratory Research

Article Title: HGF alleviates septic endothelial injury by inhibiting pyroptosis via the mTOR signalling pathway

doi: 10.1186/s12931-020-01480-3

Figure Lengend Snippet: HGF ameliorated endothelial cell pyroptosis by promoting mTOR signalling. a EA.hy926 cells were treated as described, and images were captured from randomly selected fields of view by bright field microscopy to assess the morphological changes characteristic of pyroptosis; scale bar = 50 μm; b Relative paracellular permeability of single-layer EA.hy926 cells exposed to different treatments was measured by FITC-dextran; c IL-1β in the supernatant of treated EA.hy926 cells was measured by ELISA; d LDH release assay was performed to measure pyroptosis of treated EA.hy926 cells; e CASP-1, CASP-1-P10, GSDMD and GSDMD-N in the treated EA.hy926 cell homogenates were measured by Western blot; n = 3, * P < 0.05 compared with the tLPS group; # P < 0.05 compared with the tLPS+HGF group; PHA: PHA-665752, RAPA: rapamycin

Article Snippet: The mouse monoclonal antibodies against gasdermin D (GSDMD) (sc-393,581) and β-actin (sc-517,582) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Microscopy, Permeability, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot

Journal: Stem Cells Translational Medicine

Article Title: A replicating stem‐like cell that contributes to bone morphogenetic protein 2‐induced heterotopic bone formation

doi: 10.1002/sctm.20-0378

Figure Lengend Snippet: GLAST‐TR is expressed in chondrocytes and osteoblasts during HBF. A, Representative photomicrographs of GLAST‐TR + expression in tissue during HBF. GlastTR, Neurofilament H (NF), and platelet‐endothelial cell adhesion molecule 1 (Pecam, Cd31) were detected through immunohistochemical staining during HBF and are depicted for days 0 (panels a–e) and 4 after induction with GLAST‐TR + cells first observed at day 4 (panels f–j). In addition, GLAST‐TR + Sox 9 + chondrocytes were observed in newly forming cartilage in tissues isolated 6 days after induction of bone formation (panels k–n). Furthermore, by day 14, GLAST‐TR + Sp7 + osteoblasts were observed associated with bone matrix (panels o–r). B, Tissues undergoing cartilage formation 6 days after BMP2 induction in the presence of tamoxifen were analyzed and Sox9 + chondrocytes and Sox9 + GLAST‐TR + dual positive chondrocytes from six samples were quantified using image J. The resultant percentages of GLAST‐TR + chondrocytes (Sox9 + ) are depicted graphically (symbols), the horizontal bar and error bars represent the group mean and SEM, respectively. Similarly, the percentage of GLAST‐TR + osteoblasts was calculated from six samples. Osteoblasts were defined as Sp7 + cells associated with bone matrix. Symbols on the graph are actual data points and the horizontal line depicts the group mean. The error bars represent SEM. C, Results of flow cytometry analysis to quantify the number of GLAST‐TR + cells in the hind limb. Symbols are the actual data points and a horizontal line represents the mean per group. The vertical line depicts the SEM

Article Snippet: Primary antibodies used were as follows: Sp7 (Santa Cruz Biotechnology, Inc, Dallas, Texas, monoclonal sc‐393 325), Sox 9 (Abcam, monoclonal, ab185966), Birc5 (Abcam, polyclonal, ab469), and Ki67 (Abcam, ab15580).

Techniques: Expressing, Immunohistochemical staining, Staining, Isolation, Flow Cytometry

Journal: Stem Cells Translational Medicine

Article Title: A replicating stem‐like cell that contributes to bone morphogenetic protein 2‐induced heterotopic bone formation

doi: 10.1002/sctm.20-0378

Figure Lengend Snippet: Representative photomicrographs of immunostaining confirming the existence of RSCs and COPs in tissues isolated from HBF. Tissues were isolated 10 days after BMP2‐induced HBF, fixed using sucrose, snap frozen, serial sectioned, and immunostained. A, Representative photomicrograph of Ki67(magenta), GLAST‐TR (red), Birc5 (green). White arrows show triple positive cells; green arrows show cells positive only for Birc5; red arrows show cells positive only for Glast‐TR and Birc5; purple arrows show cells positive only for Ki67. B, Representative photomicrograph of Sox9 (green), GLAST‐TR (red), Sp7 (magenta), and the merge. White arrows show triple positive cells; green arrows show cells positive only for Sox9; magenta arrows show cells positive only for Glast‐TR and Sp7

Article Snippet: Primary antibodies used were as follows: Sp7 (Santa Cruz Biotechnology, Inc, Dallas, Texas, monoclonal sc‐393 325), Sox 9 (Abcam, monoclonal, ab185966), Birc5 (Abcam, polyclonal, ab469), and Ki67 (Abcam, ab15580).

Techniques: Immunostaining, Isolation