Journal: In Vitro Cellular & Developmental Biology. Animal

Article Title: Inflammasome involvement in CS-induced damage in HaCaT keratinocytes

doi: 10.1007/s11626-022-00658-x

Figure Lengend Snippet: ( A ) Immunocytochemical analysis of NF-κB in HaCaT cells exposed to CS for 15 min compared to ctrl samples (air), 0, 1, and 3 h (T0, T1, and T3) upon the end of 15-min CS exposure. Green staining represents NF-κB, while DAPI fluorescent stain ( blue staining ) was used to counterstain the cell nuclei. Pictures were taken at 40 × magnification; scale bar = 20 μm. Fluorescence intensity for NF-κB was measured using ImageJ software. ( B ) Protein expression levels of NF-κB in cytosol ( upper panel ) and nuclei ( below panel ) in HaCaT cells exposed to CS for 15 min and collected at the selected time points starting from the end of the CS insult, with relative expression level quantification. α-Tubulin and lamin A/C were used as loading control for cytoplasm and nuclei fractions respectively. ImageJ software was used for the analysis. ( C ) Protein expression levels of NLRP3 in HaCaT samples exposed to CS and collected 0, 1, 3, and 6 h post 15 min of CS exposure. The NLRP3 expression level was quantified using ImageJ software and β-actin was used as internal control. ( D ) ASC oligomer levels in HaCaT samples 6 h (T6) post 15-min CS exposure with relative expression level quantification. β-Actin was used as internal control. ( E ) Protein expression levels of caspase-1 0, 1, 3, and 6 h (T0, T1, T3, and T6) after 15 min of CS exposure in HaCaT cells. The upper panel displays the quantification of the ratio between active caspase-1 and pro-caspase-1; ImageJ software was used for the analysis. Data are the results of the averages of at least three different experiments, * p < 0.05 by one-way ANOVA

Article Snippet: Cells were then permeabilized for 10 min with 0.4% of Triton X-100 in PBS and then blocked in PBS containing 1% BSA at RT for 1 h. Coverslips were then overnight incubated at 4 °C with primary antibodies diluted in 0.25% BSA in PBS-T:4-HNE (AB5605, Merck, Darmstadt, DE) 1:200, NF-κB p65 (NB100-56712SS, Bio-Techne, Minneapolis, MN) 1:400, ASC (cat. NBP1-78,977, NovusBio, Centennial, CO) 1:100, and NALP1 (B-2) (cat. sc-166368, Santa Cruz, Dallas, TX) 1:50.

Techniques: Staining, Fluorescence, Software, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated CST5 inhibits bone resorption and activation of osteoclasts in rat models of osteoporosis via suppression of the NF‐κB pathway

doi: 10.1111/jcmm.14552

Figure Lengend Snippet: Primers used for RT‐qPCR analysis

Article Snippet: The membrane was blocked with 5% skim milk at 37°C for 1 hour and then incubated with the rabbit anti‐rat monoclonal antibodies CST5 (MAB1202, Bio‐Techne China Co., Ltd.), p65 (NB100‐2176, Bio‐Techne China Co., Ltd.), IKB (NBP2‐23901, Bio‐Techne China Co., Ltd.), receptor activator of nuclear factor‐κB (RANK; ab200369, Abcam Inc), receptor activator of nuclear factor‐κB ligand (RANKL; ab9957, Abcam Inc), osteoprotegerin (OPG; ab73400, Abcam Inc) and β‐actin (ab8226, Abcam Inc) at 4°C overnight.

Techniques: Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated CST5 inhibits bone resorption and activation of osteoclasts in rat models of osteoporosis via suppression of the NF‐κB pathway

doi: 10.1111/jcmm.14552

Figure Lengend Snippet: CST5 inhibits the NF‐κB pathway in OP rats. A, mRNA expression of CST5, RANKL, RANK, OPG, p65 and IKB in each group evaluated by RT‐qPCR; B, the protein bands of CST5, RANKL, RANK, OPG, p65, IKB and β‐actin in each group; C, protein level of CST5, RANKL, RANK, OPG, p65 and IKB in each group assessed by Western blot analysis; *, P < .05 vs the normal group; #, P < .05 vs the OP group; n = 10; the experimental data were expressed as mean ± SD; the one‐way ANOVA was applied for statistical analysis; the samples were collected from femoral tissues of rats. ANOVA, analysis of variance; CST5, cystatin D; NC, negative control; NF‐κB, nuclear factor‐κB; OP, osteoporosis; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor‐κB; RANKL, receptor activator of nuclear factor‐κB ligand; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction

Article Snippet: The membrane was blocked with 5% skim milk at 37°C for 1 hour and then incubated with the rabbit anti‐rat monoclonal antibodies CST5 (MAB1202, Bio‐Techne China Co., Ltd.), p65 (NB100‐2176, Bio‐Techne China Co., Ltd.), IKB (NBP2‐23901, Bio‐Techne China Co., Ltd.), receptor activator of nuclear factor‐κB (RANK; ab200369, Abcam Inc), receptor activator of nuclear factor‐κB ligand (RANKL; ab9957, Abcam Inc), osteoprotegerin (OPG; ab73400, Abcam Inc) and β‐actin (ab8226, Abcam Inc) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated CST5 inhibits bone resorption and activation of osteoclasts in rat models of osteoporosis via suppression of the NF‐κB pathway

doi: 10.1111/jcmm.14552

Figure Lengend Snippet: CST5 suppresses the NF‐κB pathway to inhibit osteoclastic bone absorption in OP rats. A, mRNA expression of CST5, RANKL, RANK, OPG, p65 and IKB determined by RT‐qPCR; B, the grey values of CST5, RANKL, RANK, OPG, p65, IKB and β‐actin protein bands; C, protein level of CST5, RANKL, RANK, OPG, p65 and IKB measured by Western blot analysis; *, P < .05 vs the blank group; n = 10; the experimental data were expressed as mean ± SD; the one‐way ANOVA was performed for statistical analysis; the samples were collected from osteoclast of rats. ANOVA, analysis of variance; CST5, cystatin D; NC, negative control; NF‐κB, nuclear factor‐κB; OP, osteoporosis; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor‐κB; RANKL, receptor activator of nuclear factor‐κB ligand; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction

Article Snippet: The membrane was blocked with 5% skim milk at 37°C for 1 hour and then incubated with the rabbit anti‐rat monoclonal antibodies CST5 (MAB1202, Bio‐Techne China Co., Ltd.), p65 (NB100‐2176, Bio‐Techne China Co., Ltd.), IKB (NBP2‐23901, Bio‐Techne China Co., Ltd.), receptor activator of nuclear factor‐κB (RANK; ab200369, Abcam Inc), receptor activator of nuclear factor‐κB ligand (RANKL; ab9957, Abcam Inc), osteoprotegerin (OPG; ab73400, Abcam Inc) and β‐actin (ab8226, Abcam Inc) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Real-time Polymerase Chain Reaction