Journal: International Journal of Molecular Sciences

Article Title: Enhanced Anti-Cancer Effects of Conditioned Medium from Hypoxic Human Adult Dermal Fibroblasts on Cervical Cancer Cells

doi: 10.3390/ijms23095134

Figure Lengend Snippet: Nodes and edges in the PPIs of intracellular proteins up- and down-regulated (> 2-fold) in HeLa cells treated with H-CM compared with N-CM.

Article Snippet: The membranes were blocked with 4% skim milk for 2 h at room temperature and then incubated with rabbit anti-human CD19 (1:3000 dilution; cat. no. CSB-RA780821A0HU; CUSABIO, Wuhan, China), CHEK1 (1:3000 dilution; cat. no. CSB-RA176809A0HU; CUSABIO), ESR1 (1:3000 dilution; cat. no. CSB-PA11399A0Rb; CUSABIO), LCK (1:3000 dilution; cat. no. CSB-PA009798; CUSABIO), MCL1 (1:3000 dilution; cat. no. CSB-PA03829A0Rb; CUSABIO), PCNA (1:3000 dilution; cat. no. CSB-PA-208009; CUSABIO), POLA1 (1:3000 dilution; cat. no. CSB-PA002170; CUSABIO), TBP (1:3000 dilution; cat. no. CSB-RA821481A0HU; CUSABIO), or TNF antibody (1:3000 dilution; cat. no. CSB-PA07427A0Rb; CUSABIO) and mouse anti-human ꞵ-actin antibody (1:10,000 dilution; cat. no. A1978; Sigma) overnight at −4 °C.

Techniques:

Journal:

Article Title: Susceptibility to Tuberculosis: Clues from Studies with Inbred and Outbred New Zealand White Rabbits

doi: 10.1128/IAI.72.3.1700-1705.2004

Figure Lengend Snippet: Mean TNF-α production by thioglycolate-elicited peritoneal macrophages from outbred (black bars; n = 3) and inbred (grey bars; n = 3) rabbits, after a 24-h incubation with LPS or each of the three strains of virulent M. tuberculosis. Bars represent standard error. P values of <0.05 are marked by an asterisk.

Article Snippet: Flat-bottom 96-well plates (Nalge Nunc International, Rochester, N.Y.) were coated with goat anti-rabbit TNF-α polyclonal antibody (8 μg/ml; Becton Dickinson, San Diego, Calif.).

Techniques: Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Salt-Sensitive Hypertension Induces Osteoclastogenesis and Bone Resorption via Upregulation of Angiotensin II Type 1 Receptor Expression in Osteoblasts

doi: 10.3389/fcell.2022.816764

Figure Lengend Snippet: SSHTN mice showed increased TNF-α and activation of bone RAS in vivo . (A) IL-1β mRNA expression levels in normal control and SSHTN mice. (B) TNF-α mRNA expression levels in normal control and SSHTN mice. (C) AGTR1 mRNA expression levels in normal control and SSHTN mice. (D) ACE mRNA expression levels in normal control and SSHTN mice. IL-1β, TNF-α, AGTR1, and ACE mRNA levels were measured by real-time RT-PCR. Total RNA was isolated from the tibiae of normal control and SSHTN mice. Expression levels of IL-1β, TNF-α, AGTR1, and ACE mRNA were normalized relative to GAPDH ( n = 4). (E) Western blot analysis bands showing the expressions of TNF-α and AGTR1 protein in the distal femur of normal control and SSHTN mice. (F) Relative protein expression of TNF-α and AGTR1 were measured using ImageJ software ( n = 4). β-Actin was used as a loading control. (G) Serum levels of TNF-α was determined using ELISA MAX Standard Set Mouse TNF-α. Serum samples were collected from normal control and SSHTN mice ( n = 4).

Article Snippet: The membranes were blocked in Block-Ace (DS Pharma Biomedical, Osaka, Japan) for 1 h at room temperature and incubated with antibodies against phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP, phospho-SAPK/JNK (Thr183/Tyr185) (98F2), phospho-p44/42MAPK (ERK1/2) (Thr202/Tyr204), phospho-IκBα (Ser32) (14D4) (monoclonal rabbit IgG, 1:1,000; Cell Signaling Technology, Danvers, MA, United States), AGTR1 (polyclonal rabbit IgG, 1:1,000; Proteintech Group, Inc., Chicago, IL, United States), TNF-α (polyclonal rabbit IgG, 1:500; GTX110520, Funakoshi, Japan), and β-actin (monoclonal mouse IgG, 1:1,000; Sigma-Aldrich) overnight at 4°C.

Techniques: Activation Assay, In Vivo, Expressing, Quantitative RT-PCR, Isolation, Western Blot, Software, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Salt-Sensitive Hypertension Induces Osteoclastogenesis and Bone Resorption via Upregulation of Angiotensin II Type 1 Receptor Expression in Osteoblasts

doi: 10.3389/fcell.2022.816764

Figure Lengend Snippet: TNF-α had no effect on AGTR1 mRNA expression in osteoclast precursors but increased AGTR1 mRNA expression in osteoblasts through p38 activation. (A) AGTR1 mRNA expression level in TNF-α pretreated osteoclast precursors and (B) osteoblasts for 24 h. AGTR1 mRNA expression level was determined by real-time RT-PCR. The levels of AGTR1 mRNA expression were normalized relative to GAPDH ( n = 4). (C) Osteoblasts were incubated with TNF-α for 0, 5, 15, 30, or 60 min. Cells were lysed and analyzed by Western blotting using antibodies to phospho-ERK1/2, phospho-p38, phospho-JNK, and phospho-IκBα. β-Actin was used as a loading control. (D) Inhibition of p38 signaling prevented induction of AGTR1 protein expression by TNF-α in vitro . Osteoblasts were preincubated with or without MAPK and NF-κB inhibitors and then treated with TNF-α for 24 h. Cells were lysed and analyzed by Western blotting using an antibody to AGTR1. (E) Relative protein expression of AGTR1 were measured using ImageJ software ( n = 3). β-Actin was used as a loading control.

Article Snippet: The membranes were blocked in Block-Ace (DS Pharma Biomedical, Osaka, Japan) for 1 h at room temperature and incubated with antibodies against phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP, phospho-SAPK/JNK (Thr183/Tyr185) (98F2), phospho-p44/42MAPK (ERK1/2) (Thr202/Tyr204), phospho-IκBα (Ser32) (14D4) (monoclonal rabbit IgG, 1:1,000; Cell Signaling Technology, Danvers, MA, United States), AGTR1 (polyclonal rabbit IgG, 1:1,000; Proteintech Group, Inc., Chicago, IL, United States), TNF-α (polyclonal rabbit IgG, 1:500; GTX110520, Funakoshi, Japan), and β-actin (monoclonal mouse IgG, 1:1,000; Sigma-Aldrich) overnight at 4°C.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Incubation, Western Blot, Inhibition, In Vitro, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Salt-Sensitive Hypertension Induces Osteoclastogenesis and Bone Resorption via Upregulation of Angiotensin II Type 1 Receptor Expression in Osteoblasts

doi: 10.3389/fcell.2022.816764

Figure Lengend Snippet: Angiotensin II enhanced the TNF-α-induced increase in RANKL/OPG ratio in osteoblasts and enhanced osteoclastogenesis in TNF-α-primed osteoblast and osteoclast precursor co-culture. (A) RANKL mRNA expression level in osteoblasts. (B) OPG mRNA expression level in osteoblasts. (C) RANKL/OPG mRNA expression levels in osteoblasts. RANKL and OPG mRNA expression levels were determined by real-time RT-PCR and were normalized relative to GAPDH ( n = 3). (D) Micrographs and (E) number of large TRAP-positive cells in co-cultures of TNF-α-primed or non-primed osteoblasts and osteoclast precursors treated with PBS, 1.25(OH) 2 D 3 + PGE 2 , 1.25(OH) 2 D 3 + PGE 2 + angiotensin II, and 1.25(OH) 2 D 3 + PGE 2 + angiotensin II + angiotensin II type 1 receptor blocker. Scale bar = 200 μm ( n = 4).

Article Snippet: The membranes were blocked in Block-Ace (DS Pharma Biomedical, Osaka, Japan) for 1 h at room temperature and incubated with antibodies against phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP, phospho-SAPK/JNK (Thr183/Tyr185) (98F2), phospho-p44/42MAPK (ERK1/2) (Thr202/Tyr204), phospho-IκBα (Ser32) (14D4) (monoclonal rabbit IgG, 1:1,000; Cell Signaling Technology, Danvers, MA, United States), AGTR1 (polyclonal rabbit IgG, 1:1,000; Proteintech Group, Inc., Chicago, IL, United States), TNF-α (polyclonal rabbit IgG, 1:500; GTX110520, Funakoshi, Japan), and β-actin (monoclonal mouse IgG, 1:1,000; Sigma-Aldrich) overnight at 4°C.

Techniques: Co-Culture Assay, Expressing, Quantitative RT-PCR