Journal: Scientific Reports

Article Title: CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

doi: 10.1038/s41598-021-90271-x

Figure Lengend Snippet: Immunofluorescence analysis of persistent expression of fusion-regulatory proteins in CD13-deficient multinucleated OC. Expression of the fusion regulatory proteins dynamin ( a , d ), DCST1 ( b , e ) and CD9 ( c , f ) is maintained in CD13 KO multinucleated osteoclasts but not in WT cells (**). High levels of dynamin co-localize with actin and DCST1 in OCPs (*), imaged using Zeiss LSM 880 confocal fluorescence microscope and analyzed by Zeiss Zen 2.0 Pro blue edition software ( https://www.zeiss.com/content/dam/Microscopy/Downloads/Pdf/FAQs/zen2-blue-edition_installation-guide.pdf ). Scale bar; 10 µm. Data represents average of three independent experiments. N = 3/genotype. Dynamin, CD9, DCST1; green. Phalloidin; red. Magnification ×63 oil.

Article Snippet: Surface expression of fusion promoting proteins was performed with goat anti-rabbit dynamin2-Alexa 488 (ProSci; 61-336), rabbit anti-mouse DCST1-Alexa fluor 350 (Bioss Inc; bs-8250R-A350), and rat anti-mouse CD9-APC (Biolegend, Clone MZ3; 124811) using BD LSRII-A and analyzed by FlowJo version 9.9 software ( https://www.flowjo.com/ ).

Techniques: Immunofluorescence, Expressing, Fluorescence, Microscopy, Software

Journal: Scientific Reports

Article Title: CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

doi: 10.1038/s41598-021-90271-x

Figure Lengend Snippet: Immunoblot analysis of sustained expression of fusion-regulatory proteins in CD13-deficient OC over time. Expression of the fusion-promoting proteins dynamin, DC-STAMP (DCST1) and CD9 is aberrantly sustained in CD13 KO but not in WT multinucleated osteoclasts ( a , b ) in presence of M-CSF and RANKL. A cropped image is presented, see Supplementary Fig. for full-length blots and Supplementary Fig. for cropped replicates. ( c , d ). CD13 expression is unaltered in BM-derived OCs in response to M-CSF and RANKL stimulation over time. Blots were imaged by ChemiDoc Imaging system version 3.0.1 ( https://www.bio-rad.com/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15 ) (Biorad). A cropped image is presented, see Supplementary Fig. for full-length blots and cropped replicates. Data represents average of two isolates. N = 3/genotype. **p < 0.01, *p < 0.05.

Article Snippet: Surface expression of fusion promoting proteins was performed with goat anti-rabbit dynamin2-Alexa 488 (ProSci; 61-336), rabbit anti-mouse DCST1-Alexa fluor 350 (Bioss Inc; bs-8250R-A350), and rat anti-mouse CD9-APC (Biolegend, Clone MZ3; 124811) using BD LSRII-A and analyzed by FlowJo version 9.9 software ( https://www.flowjo.com/ ).

Techniques: Western Blot, Expressing, Derivative Assay, Imaging

Journal: Scientific Reports

Article Title: CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

doi: 10.1038/s41598-021-90271-x

Figure Lengend Snippet: Fusion-regulatory proteins, dynamin and DCST1 are regulated by a CD13-dependent post transcriptional mechanism. ( a ) Quantitative RT-PCR analysis of fusion regulatory transcripts normalized to GAPDH in flow sorted mouse BM cells stimulated with M-CSF and RANKL over indicated time. Expression of the genes regulating osteoclast fusion -dynamin 2 (DNM2), DCST1, CD9 and CD81 are highly induced upon M-CSF and RANKL over time but was equivalent between genotypes. All data was analyzed using CFX Manager version 3.1 (( https://www.bio-rad.com/en-us/sku/1845000-cfx-manager-software?ID=1845000 ) (Biorad). Data represents average of two independent experiments. N = 3/genotype. ( b – c ) Dynamin and DCST1 protein stability are enhanced in absence of CD13. Immunoblot analysis of dynamin and DCST1 of WT and CD13 KO BM-derived OC treated with cycloheximide (CHX) for indicated time. Blots were imaged by ChemiDoc Imaging system version 3.0.1 ( https://www.bio-rad.com/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15 ) (Biorad). A cropped image is presented, see Supplementary Fig. for full-length blots and cropped replicates. Data represents average of two isolates. N = 3/genotype. **p < 0.01, *p < 0.05.

Article Snippet: Surface expression of fusion promoting proteins was performed with goat anti-rabbit dynamin2-Alexa 488 (ProSci; 61-336), rabbit anti-mouse DCST1-Alexa fluor 350 (Bioss Inc; bs-8250R-A350), and rat anti-mouse CD9-APC (Biolegend, Clone MZ3; 124811) using BD LSRII-A and analyzed by FlowJo version 9.9 software ( https://www.flowjo.com/ ).

Techniques: Quantitative RT-PCR, Expressing, Software, Western Blot, Derivative Assay, Imaging

Journal: Genes

Article Title: In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls

doi: 10.3390/genes13030551

Figure Lengend Snippet: The influence of the DNA probes on the expression of AIM2 gene in HSFs. ( A ) Change in the RNA AIM2 in HSFs (n = 10) in the presence of DNA probes (50 ng/mL. The cultivation time and the DNA probes are indicated on the graph. Sz-HSFs did not differ from hc-HSFs in terms of the RNA AIM2 levels. ( B ) The most typical examples of the AIM2 analysis with FCA in HSFs. The data from the device are given for hc-HSF5 (1 h). EEA1: green color; background: black color. ( C ) Index AIM2: the values of the medians of FL-AIM2, normalized to the maximum signal value in the sample (n = 180). Average values for three measurements and standard deviation are given. ( D ) Analysis of relative changes in AIM2 levels in the presence of DNA probes compared to the control. Figures indicate the ratio (AIM2 probe DNA—AIM2 control)/AIM2 control. Green color—no significant differences with the control ( p > 0.01), red—increased protein content in the presence of the DNA probe ( p < 0.01) and blue—decreased protein content ( p < 0.01). ( E ) Changes in AIM2 level in hc-HSFs (n = 5). ( F ) Changes in the ratios RNA TLR9 to RNA AIM2 and TLR9 to AIM2 in samples of HSFs (n = 10). ( G ) Visualization of AIM2 (PE) in HSFs. An example is given for the sz-cfDNA probe and the sz-HSF1 (1 h).

Article Snippet: When stained with the conjugated antibodies FITC-HMGB1 (NB100-2322F Novus Biologicals, LLC, Centennial, CO, USA) and AF350-AIM2 (bs-5986r-a350 Bioss Inc., Woburn, MA, USA), cells were incubated for 3 h at 4 °C in the dark at room temperature and washed thrice with 0.5% BSA-PBS.

Techniques: Expressing, Standard Deviation

Journal: Genes

Article Title: In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls

doi: 10.3390/genes13030551

Figure Lengend Snippet: Spearman’s rank correlation (Rs and p -value) for the protein levels in HSFs incubated with DNA probes.

Article Snippet: When stained with the conjugated antibodies FITC-HMGB1 (NB100-2322F Novus Biologicals, LLC, Centennial, CO, USA) and AF350-AIM2 (bs-5986r-a350 Bioss Inc., Woburn, MA, USA), cells were incubated for 3 h at 4 °C in the dark at room temperature and washed thrice with 0.5% BSA-PBS.

Techniques: Incubation