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: International Journal of Molecular Sciences

Article Title: Deciphering the Role of ADAMTS6 in the Epithelial–Mesenchymal Transition of Lung Adenocarcinoma Cells

doi: 10.3390/ijms262411850

Figure Lengend Snippet: Effect of ADAMTS6 knockout on EMT. Control, parental A549 cells; AD6-KO, A549 cells with ADAMTS6 gene knockout. ( A ) Relative expression of the epithelial marker CDH1 and the mesenchymal markers CDH2 , VIM , and TWIST1 , assessed using RT-qPCR and normalized to GAPDH expression. ( B ) IF staining of vimentin (green) and DAPI staining of nuclei (blue). ( C ) IF staining of NF-κB p65 (green) and DAPI staining of nuclei (blue). p65 nuclear translocation was assessed by the nuclear to cytoplasm (Nuc/cyt) fluorescence ratio. ( D ) Cell viability after 72 h of incubation with specified concentrations of doxorubicin assessed by MTT assay. IC 50 data are presented in the table. All experiments were conducted with at least three biological replicates. Data are presented as mean ± SD. Statistical significance was determined by a two-tailed Student’s t -test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IF reagents included: rabbit anti-vimentin (ab92547), rabbit anti-SMAD2/3 (ab202445), AlexaFluor 488-conjugated goat anti-IgG (ab150077) antibodies (all from Abcam, Waltham, MA, USA), rabbit anti-p65 (A2547, ABclonal, Wuhan, China), DAPI, and Fluoromount-G (both from Thermo Fisher Scientific, Rockford, IL, USA).

Techniques: Knock-Out, Control, Gene Knockout, Expressing, Marker, Quantitative RT-PCR, Staining, Translocation Assay, Fluorescence, Incubation, MTT Assay, Two Tailed Test

Journal: Channels (Austin, Tex.)

Article Title: Identification of SLC2A3 as a prognostic indicator correlated with the NF-κB/EMT axis and immune response in head and neck squamous cell carcinoma.

doi: 10.1080/19336950.2023.2208928

Figure Lengend Snippet: Figure 5. SLC2A3 expression was associated with NF-κB, and NF-κB was associated with EMT gene signatures in TCGA-HNSC. (a) GSEA of DEGs between the low and high SLC2A3 expression groups from TCGA-HNSC for NF-κB signaling. NES: normalized enrichment score. FDR: false discovery rate. (b) Correlation between SLC2A3 and the expression of NF-κB1, NF-κB2, REL, RELA and RELB. (c) Both RELB and NF-κB2 were positively correlated with the expression of N-cad, vimentin, Snai1 and MMP9 and negatively correlated with the expression of E-cad.

Article Snippet: After transfer, the membrane was incubated with primary antibodies against SLC2A3 (R24453, Zen Bio), E-cad (bs -10,009 R, Bioss), N-cad (bs-1172 R, Bioss), Vimentin (bs-0756 R, Bioss), Snai1 (bs-1371 R, Bioss), and NFκB P65 (AP0123, ABclonal).

Techniques: Expressing

Journal: Channels (Austin, Tex.)

Article Title: Identification of SLC2A3 as a prognostic indicator correlated with the NF-κB/EMT axis and immune response in head and neck squamous cell carcinoma.

doi: 10.1080/19336950.2023.2208928

Figure Lengend Snippet: Figure 9. SLC2A3 knockdown inhibited proliferation and migration and NF-κB/EMT-related gene expression in FaDu and CNE-2 cell lines. (a) SLC2A3 mRNA expression in adjacent normal tissues (ANT) and HNSC samples. (b) Cell proliferation and (c) migration were suppressed upon SLC2A3 knockdown. (d) SLC2A3 knockdown inhibited N-cad, Vimentin, and Snai1 expression but increased E-cad expression. (e) NF-κB P65 expression was decreased upon SLC2A3 knockdown. Each value represents the mean ± SD of three separate experiments. * p < 0.05, # p < 0.01.

Article Snippet: After transfer, the membrane was incubated with primary antibodies against SLC2A3 (R24453, Zen Bio), E-cad (bs -10,009 R, Bioss), N-cad (bs-1172 R, Bioss), Vimentin (bs-0756 R, Bioss), Snai1 (bs-1371 R, Bioss), and NFκB P65 (AP0123, ABclonal).

Techniques: Knockdown, Migration, Gene Expression, Expressing

Journal: Bioengineered

Article Title: C1q/tumor necrosis factor-related protein-3 (CTRP3) activated by forkhead box O4 (FOXO4) down-regulation protects retinal pericytes against high glucose-induced oxidative damage through nuclear factor erythroid 2-related factor 2 (Nrf2)/Nuclear factor-kappaB (NF-κB) signaling

doi: 10.1080/21655979.2022.2031413

Figure Lengend Snippet: CTRP3 inactivated by FOXO4 modulates the Nrf2/NF-κB signaling pathway. The protein levels of Nrf2, p-NF-κB p65 and NF-κB p65 were analyzed by Western blot. ***P < 0.001 vs. MA. ### P < 0.001 vs. HG+Ov-NC. $$$ P < 0.001 vs. HG+Ov-CTRP3+ Ov-NC. CTRP3, C1q/tumor necrosis factor-related protein-3. HG, high glucose. MA, mannitol. FOXO4, forkhead box O4. Nrf2, nuclear factor erythroid 2-related factor 2. p-NF-κB p65, phosphorylated-Nuclear factor-kappaB p65. NF-κB p65, Nuclear factor-kappaB p65.

Article Snippet: The membranes were impeded by 5% nonfat milk and incubated with the following primary antibodies: anti-CTRP3 (Abcam, ab36870), anti-B cell lymphoma-2 (Bcl-2) (Abcam, 1:1000, ab32124), anti-BCL-2 associated X (Bax) (Abcam, 1:1000, ab32503), anti-cleaved caspase 3 (Abcam, 1:500, ab32042), anti-caspase 3 (Abcam, 1:5000, ab32351), anti-cleaved Poly (ADP-ribose) polymerase (PARP) (Abcam, 1:1000, ab32064), PARP (Abcam, 1:1000, ab191217), anti-FOXO4 (Abcam, 1:1000, ab128908), anti-Nrf2 (Abcam, 1:1000, ab62352), anti- phosphorylated (p-)NF-κB p65 (Abcam, 1:1000, ab76302), anti-NF-κB p65 (Abcam, 1:1000, ab32536) and anti-GAPDH (Abcam, 1:2500, ab9485) at 4°C overnight.

Techniques: Western Blot