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: The Prostate

Article Title: RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model

doi: 10.1002/pros.23925

Figure Lengend Snippet: RELA mediates IL-1 repression of AR. A, RT-qPCR and B, Western blot analysis and densitometry were performed for LNCaP, C4-2, and C4-2B cells transfected with 70 nM of nontargeting or RELA siRNA (Dharmacon) for 1 day followed by treatment with vehicle control (Veh), IL-1α, IL-1β for 2 days. LNCaP cells were treated with 0.5 ng/mL IL-1, and C4-2 and C4-2B cells were treated with 25 ng/mL IL-1. RELA siRNA attenuates IL-1 downregulation of AR and PSA mRNA and protein, and attenuates IL-1 upregulation of SOD2 mRNA and protein. mRNA fold change is normalized to the vehicle control. Error bars indicate ±SD of three biological replicates; *P ≤ .05, **P ≤ .005, ***P ≤ .0005. AR, androgen receptor; IL, interleukin; mRNA, messenger RNA; PSA, prostate-specific antigen; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA; SOD2, superoxide dismutase 2

Article Snippet: The following siRNA concentrations were used: 70 nM nontargeting siRNA (D-001206-13-05; Dharmacon), RELA siRNA (M-003533-02-0005; Dharmacon), NFKB1 siRNA (M-003520-01-0005; Dharmacon), or p62 siRNA (M-010230-00-0005; Dharmacon); 100 nM nontargeting siRNA, RELB siRNA (M-004767-02-0005; Dharmacon), c-REL siRNA (M-004768-01-0005; Dharmacon), or NFKB2 siRNA (M-003918-02–0005; Dharmacon); 70 nM nontargeting siRNA (SR30004; Origene) or RELA siRNA (SR321602; Origene).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: The Prostate

Article Title: RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model

doi: 10.1002/pros.23925

Figure Lengend Snippet: RELA siRNA restores nuclear AR accumulation in IL-1-treated PCa cells. C4-2 and C4-2B cells transfected with 70 nM of nontargeting or RELA siRNA (Dharmacon) for 1 day followed by treatment with vehicle control (Veh), 25 ng/mL IL-1α, or 25 ng/mL IL-1β for 2 days. A, Cells were fixed and immunostained for AR (Texas Red) and cell nuclei (DAPI, blue) and imaged at ×20 magnification. AR/DAPI merge images are shown for C4-2 (left) and C4-2B (right). B, The ratio (AR/DAPI) of the number of cells accumulating AR to the number of DAPI-stained cells was calculated for five microscopy fields at ×10 magnification, n > 300 cells per field. AR/DAPI ratio was normalized to vehicle control siRNA. RELA siRNA restores AR nuclear accumulation in IL-1-treated cells. Error bars indicate ±SD of five microscopy fields; ***P ≤ .0005. AR, androgen receptor; DAPI, 4′,6-diamidino-2-phenylindole; IL, interleukin; SD, standard deviation; siRNA, small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The following siRNA concentrations were used: 70 nM nontargeting siRNA (D-001206-13-05; Dharmacon), RELA siRNA (M-003533-02-0005; Dharmacon), NFKB1 siRNA (M-003520-01-0005; Dharmacon), or p62 siRNA (M-010230-00-0005; Dharmacon); 100 nM nontargeting siRNA, RELB siRNA (M-004767-02-0005; Dharmacon), c-REL siRNA (M-004768-01-0005; Dharmacon), or NFKB2 siRNA (M-003918-02–0005; Dharmacon); 70 nM nontargeting siRNA (SR30004; Origene) or RELA siRNA (SR321602; Origene).

Techniques: Transfection, Staining, Microscopy, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Heme oxygenase 1‑overexpressing bone marrow mesenchymal stem cell‑derived exosomes suppress interleukin‑1 beta‑induced apoptosis and aging of nucleus pulposus cells

doi: 10.3892/mmr.2025.13481

Figure Lengend Snippet: Activation of the NF-κB signalling in NP cells induced by IL-1β was repressed after Exo-HO-1 treatment. Western blotting was performed to detect p-p65 and/or p65 protein levels in NP cells with different treatments (control, IL-1β, IL-1β + Exo and IL-1β + Exo-HO-1) as well as in the nuclear (N) and cytoplasmic (C) fractions of the cells. Histone H3 was used as an internal reference for the nucleus. ***P<0.001 vs. control; # P<0.05, ## P<0.01 and ### P<0.001 vs. IL-1β. NP, nucleus pulposus; IL, interleukin; Exo, exosomes; HO-1, heme oxygenase 1; p-phosphorylated.

Article Snippet: After blocking with a rapid blocking solution (Beyotime Institute of Biotechnology) at 4°C overnight, the membranes were incubated with appropriate primary antibodies including anti-HO-1 (cat. no. GTX637432; 1:2,000; GeneTex, Inc.), anti-TGS101 (cat. no. A01233-2, 0.25 μg/ml; Boster Bio), anti-CD63 (FNab01490; 1:1,000; Fine Biotech Co., Ltd, Wuhan, China) and anti-Cainexin (cat. no. GTX109669; 1:5,000; GeneTex, Inc.), Bcl-2 (cat. no. FNab00839; 1:1,000; Wuhan Fine Biotech Co., Ltd.), Bax (cat. no. FNab00810; 1:2,000; Wuhan Fine Biotech Co., Ltd.), cleaved caspase 3 (cat. no. MBS9410752; 1:1,000; MyBioSource, Inc.), anti-p65 (cat. no. PB9324; 0.5 μg/ml; Boster Bio), anti-p-p65 (cat. no. A00284S468-2; 1:2,000; Boster Bio), anti-GAPDH (cat. no. H00227; 1:5,000; Boster Bio) and anti-Histone H3 (cat. no. M12477-9; 1:1,000; Boster Bio) antibodies.

Techniques: Activation Assay, Western Blot, Control

Journal: International Journal of Nanomedicine

Article Title: Iron oxide magnetic nanoparticles combined with actein suppress non-small-cell lung cancer growth in a p53-dependent manner

doi: 10.2147/IJN.S127549

Figure Lengend Snippet: Fe 3 O 4 MNPs and AT induced apoptosis in NSCLC cells through caspase 3 activation. Notes: ( A ) Left: A549 cells were given AT, MNPs, or a combination of the two at the concentrations indicated for 24 hours. Total protein was extracted and subjected to immunoblot analysis for cleaved caspase 8, caspase 9, and caspase 3 detection. GAPDH was used as loading control. Right: quantification of Western blot analysis. ( B ) Left: H1975 cells were treated with AT, MNPs, or AT-MNP combination for 24 hours. Whole-cell protein was extracted and examined via immunoblot analysis for cleaved caspase 8, caspase 9 and caspase 3 determination. GAPDH was used as loading control. Right: quantification of Western blot analysis. ( C ) Caspase 3 (left) and caspase 9 (right) activation was determined in A549 cells administered AT and MNPs alone or a combination thereof in the absence or presence of caspase 3 or caspase 9 inhibitor for 24 hours. ( D ) Caspase 3 (left) and caspase 9 (right) activation was determined in H1975 cells treated with AT and MNPs alone or a combination thereof in the absence or presence of caspase 3 or caspase 9 inhibitor for 24 hours. Values are expressed as means ± standard error of mean. * P <0.05, ** P <0.01, and *** P <0.001 vs Con group; # P <0.05, ## P <0.01, and ### P <0.001. Analysis of variance and Dunnett’s analysis were included to compare the averages of multiple groups. Abbreviations: MNPs, magnetic nanoparticles; AT, actein; NSCLC, non-small-cell lung cancer; Con, control; NS, not significant.

Article Snippet: Rabbit anti-caspase 9 , 1:1,000 , Abcam (ab32539).

Techniques: Activation Assay, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Iron oxide magnetic nanoparticles combined with actein suppress non-small-cell lung cancer growth in a p53-dependent manner

doi: 10.2147/IJN.S127549

Figure Lengend Snippet: Working model of the effect of Fe 3 O 4 magnetic nanoparticle and actein combination on NSCLC cells. Notes: Fe 3 O 4 magnetic nanoparticle and actein combination treatment induces apoptosis in NSCLC cells in a p53-dependent manner. p53 activation results in TRAIL and its DR4 and DR5 contributing to caspase 8 activity through FADD stimulation. Further, p53 activity reduces Bcl2 and BclXL expression, and promotes Bax and Bad expression levels. Therefore, the ratio of Bax:Bcl2 is increased, which leads to activation of caspase 9 and caspase 3. Consequently, apoptosis in NSCLC cells is induced, inhibiting NSCLC development. Abbreviation: NSCLC, non-small-cell lung cancer.

Article Snippet: Rabbit anti-caspase 9 , 1:1,000 , Abcam (ab32539).

Techniques: Activation Assay, Activity Assay, Expressing

Journal: Translational Cancer Research

Article Title: Extracellular vesicles isolated from curcumin-medium weakened RKO cell proliferation and migration

doi: 10.21037/tcr-24-98

Figure Lengend Snippet: Curcumin suppressed the expression of NF-κB p65 in RKO EVs. The expression of NF-κB p65 in RKO EVs, Cur1-RKO EVs and Cur10-RKO EVs was analyzed by western blotting, respectively. The data are presented as mean ± SD (*, P<0.05). EVs, extracellular vesicles; Cur, curcumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, no significance; SD, standard deviation.

Article Snippet: Polyclonal antibodies against proliferating cell nuclear antigen (PCNA; HY-P80268, 1:4,000), Bcl-2-related X protein (Bax) (HY-P80028, 1:4,000), NF-κB p65 (HY-P80765, 1:1,000), and Calnexin (HY-P80578, 1:1,000) were obtained from MedChemExpress, Shanghai, China.

Techniques: Expressing, Western Blot, Standard Deviation