Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Silence of lncRNA MIAT protects ATDC5 cells against lipopolysaccharides challenge via up-regulating miR-132.

doi: 10.1080/21691401.2019.1626410

Figure Lengend Snippet: Figure 4. Suppressive effect of MIAT silence on the activation of NF-jB and JNK pathways via miR-132. ATDC5 cells were co-transfected with sh-MIAT and miR-132 inhibitor and then subjected to 6 lg/mL LPS. The phosphor/total (p/t) levels of (A,B) p65, IjBa and (C,D) JNK were measured using western blot (n ¼ 3). p < .05 (ANOVA combined with Duncan post-hoc test).

Article Snippet: Anti-caspase-3 (orb378617), anti-cleavedcaspase-3 (orb106556), anti-caspase-9 (orb135175), anticleaved-caspase-9 (orb227889), anti-PARP (orb526607), anti-cleaved-PARP (orb106557), anti-IL-6 (orb6210), anti-IL-8 (orb39299), anti-TNF-a (orb475251), anti-MCP-1 (orb97456), anti-p65 (orb344389), anti-p-p65 (orb14753), anti-IjBa (orb338946), anti-p-IjBa (orb99312), anti-JNK (orb38050), anti-p-JNK (orb184488), and anti-b-actin (orb86987) all from Biorbyt (San Francisco, CA).

Techniques: Activation Assay, Transfection, Western Blot

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Brazilin blocks catabolic processes in human osteoarthritic chondrocytes via inhibition of NFKB1/p50.

doi: 10.1002/jor.24013

Figure Lengend Snippet: Figure 4. Brazilin inhibits NFKB1/p50. (a) Primary human chondrocytes (n ¼ 4 donors) were left un- treated (C), treated with 10 ng/ml IL-1b for 6 h, or pre-incubated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 6 h. NFKB1 mRNA levels were analyzed by RT-qPCR and expressed as relative quantities (mean SD) com- pared to controls. Significant differences compared to untreated cells (p < 0.05; n ¼ 4; Quade test). #Sig- nificant differences compared to cells treated only with IL-1b (p < 0.05; n ¼ 4; Quade test). (b) Primary human chondrocytes (n ¼ 4 donors) were left un- treated (C) or were treated with 10 ng/ml IL-1b for 15 min, 1 h, 4 h, 16 h, and 32 h. Protein extracts were subjected to Western blot analysis for the detection of p50, p65, and phosphorylated p65. b-Actin and a- tubulin were used as loading controls. The blot of one representative experiment is shown. (c) Primary chondrocytes (n ¼ 4) were left untreated (C), treated with 10 ng/ml IL-1b for 24 h, or pre-treated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 24 h. Protein extracts were sub- jected to Western blot analysis for the detection of p50. b-Actin was used as loading control. The blot of one representative experiment is shown. (d) Primary chondrocytes (n ¼ 4) were left untreated (C), treated with 10 ng/ml IL-1b for 1 h, or pre-treated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 1 h. Protein extracts were subjected to Western blot analysis for the detection of p65 and phosphorylated p65. a-Tubulin was used as loading control. The blot of one representative experiment is shown. (e–h) Primary chondrocytes (n ¼ 3 donors) were left untreated as controls, treated with 10 ng/ml IL-1b for 6 h, or pre-treated with 2.5, 5, and 10 mg/ml brazilin or 5, 10, and 20 mg/ml CAPE for 1 h prior to stimulation with 10 ng/ml IL-1b for 6 h. The mRNA levels of (e) IL1B, (f) TNF, (g) PTGS2, and (h) MMP13 were analyzed by RT-qPCR and expressed as percent relative to cells treated with IL-1b (100%). Significant differences compared to cells treated only with IL-1b (p < 0.05; n ¼ 3; Friedman test with many- to-one post-hoc test).

Article Snippet: After blocking, the membranes were probed for 2h with primary antibodies: Anti-NF-kB p105/p50 (1:400, ab7971, rabbit polyclonal; Abcam, Cambridge, MA), anti-NF-kB p65 (1:1,000, rabbit monoclonal; Cell Signaling Technology, Danvers, MA), anti-phospho-NF-kB p65 (Ser536, 1:1,000, rabbit monoclonal; Cell Signaling Technology), anti-a-tubulin (1:1,000, mouse monoclonal; Cell Signaling Technology) and anti-b-actin (1:5,000, mouse monoclonal; Sigma-Aldrich).

Techniques: Incubation, Quantitative RT-PCR, Western Blot, Control

Journal: Toxics

Article Title: Ferulic Acid Derivatives Ameliorate Intestine Barrier Destruction by Alleviating Inflammatory Responses in Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease

doi: 10.3390/toxics12040268

Figure Lengend Snippet: Anti-inflammatory effect of FA derivatives C1 and C1a in macrophages: ( a ) the cytotoxicity of FA, C1, and C1a was determined on J774A.1 cells and mouse peritoneal macrophages; ( b ) LPS-stimulated nitric oxide production was determined using a nitric oxide detection kit on J774A.1 cells and mouse peritoneal macrophages; ( c ) the LPS-stimulated expression of pro-inflammatory cytokines and mediators including IL-1β, IL-6, MCP-1, iNOS, and COX-2 in J774A.1 cells was analyzed by qPCR; ( d ) the LPS-stimulated activation of intracellular mediators, including iNOS and COX-2, was confirmed by Western blot. ( e ) the LPS-stimulated phosphorylation of NF-κB was assessed by ELISA. Data are means ± SEM of three independent experiments. Note: # p < 0.05 is a significant difference compared with control group; * p < 0.05 is a significant difference compared with LPS-treated group.

Article Snippet: The phosphorylation of nuclear factor (NF)-κB in LPS-stimulated J774A.1 cells was determined with an ELISA kit (#7834S; Cell Signaling Technology) according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Control