Journal: bioRxiv

Article Title: Exosomes from COVID-19 patients carry tenascin-C and fibrinogen-β in triggering inflammatory signals in distant organ cells

doi: 10.1101/2021.02.08.430369

Figure Lengend Snippet: Panel A: Lysates from COVID-19 plasma exosomes and control exosomes were subjected to Western blot analysis for TNC and FGB using specific antibodies and representative image is shown. Panel B: Dot plots for quantitative Western blot band intensities by densitometry analysis using ImageJ software (right panel) are shown (n=8 normal and n=20 COVID-19 samples). TSG101, an exosomal marker protein, was used for normalization of each sample. (**, p<0.01; ***, p<0.001). C: String analysis network module represents functional association of TNC and FGB with TLR4/NF-kB signaling. Each node represents all the proteins produced by a single protein coding gene locus. Colored node represents query proteins and first shell of interactions. Filled node shows 3D structure (known or predicted). Edges represent protein-protein associations for shared function.

Article Snippet: Cells lysates were subjected to Western blot analysis using specific antibodies to CD63 (Santa Cruz Biotechnology), TSG101 (Santa Cruz Biotechnology), tenascin (TNC) (Sigma), fibrinogen-β (FGB) (Santa Cruz Biotechnology), phospho-NF-kB p65 (Ser536) (Cell Signaling Technology, CST), NF-kB p65 (CST).

Techniques: Clinical Proteomics, Control, Western Blot, Software, Marker, Functional Assay, Produced

Journal: bioRxiv

Article Title: Exosomes from COVID-19 patients carry tenascin-C and fibrinogen-β in triggering inflammatory signals in distant organ cells

doi: 10.1101/2021.02.08.430369

Figure Lengend Snippet: Panel A. IHH were exposed to normal and COVID-19 exosomes for 48h and total RNA was isolated. Relative mRNA expression of TNF-α, IL-6 and CCL5 in cells treated with COVID-19 exosomes (n=20) or normal exosomes (n=8) were examined by qRT-PCR and represented by dot plots. 18s rRNA was used as an internal control. Small bar indicates standard error (* p<0.05). Panel B: Pearson correlation analysis among expressions of the TNF-α, IL-6 and CCL5 in the hepatocytes exposed with patient exosomes. Panels C and D: Exosomes from plasma of COVID-19 patients activates NF-kB signaling in hepatocytes. IHH (panel C) or Huh7 cells (panel D) were exposed with normal and COVID-19 exosomes for 48h and cell lysates were subjected to Western blot analysis for phospho-NF-kB p65 (Ser536), NF-kB p65 using specific antibodies. The membrane was reprobed for actin as an internal control. Right panel shows quantitative representation of Western blot band intensities. Small bar indicates standard error (*p<0.05; **p<0.01).

Article Snippet: Cells lysates were subjected to Western blot analysis using specific antibodies to CD63 (Santa Cruz Biotechnology), TSG101 (Santa Cruz Biotechnology), tenascin (TNC) (Sigma), fibrinogen-β (FGB) (Santa Cruz Biotechnology), phospho-NF-kB p65 (Ser536) (Cell Signaling Technology, CST), NF-kB p65 (CST).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Control, Clinical Proteomics, Western Blot, Membrane

Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

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: The Journal of allergy and clinical immunology

Article Title: ANKRD1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum

doi: 10.1016/j.jaci.2018.01.001

Figure Lengend Snippet: The indicated expression plasmids were transfected into 293 FT cells and incubated overnight. The cells were then harvested and co-IP experiments were performed. Flag/DDK1-tagged protein expression and HA-tagged ANKRD1 protein expression are shown in IP samples and lysates. Data presented as one of three independent experiments.

Article Snippet: Myc-DDK1-tagged RELA (RC220780), Myc-DDK1-tagged NFKB1 (RC208384), Myc-DDK1-tagged IRF7 (RC217934), Myc-DDK1-tagged ANKRD1 (RC205609) and Myc-DDK1-tagged STING were purchased from OriGene (Rockville, MD).

Techniques: Expressing, Transfection, Incubation, Co-Immunoprecipitation Assay

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