Journal: Journal of Biomedical Science

Article Title: Many but not all pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification after spinal cord injury

doi: 10.1186/s12929-026-01237-y

Figure Lengend Snippet: Expression of PRRs by mouse and human skeletal muscle cells. Expression of PRR mRNA transcripts in mouse satellite cells (SC), FAPs (FP), endothelial cells (EC) and monocyte/macrophages (MΦ) sorted from skeletal muscles. Whole mouse bone marrow (BM) cells and splenocytes (SP) were used as positive controls for these transcripts. Human PDGFRα + FAPs were sorted from muscles surrounding NHO biopsies, and CD14 + monocytes were isolated from peripheral blood from healthy donors. A TLR1, B TLR2, C TLR3, D TLR4, E TLR5, F TLR6, G TLR7, H TLR8, I TLR9, J STING1, K RIGI, L MDA5 (IFIH1), M PRK, N NOD1, O NOD2, P Dectin-1 (CLEC7A), Q Dectin-2 (Clec4n/CLEC6A), R Mincle (CLEC4E). For each gene, left hand histogram represents expression of the mouse gene in mouse cells and the right-hand histogram represents expression of the human gene in human cells. For mouse cells, relative mRNA expression was quantified relative to house-keeping gene Hprt . For human cells, values were normalized using three references genes HPRT , RPLP0 and PPIA for FAPs and ACTB , GAPDH and PPIA for CD14 + cells. Each dot represents a mouse or a human donor, bars represent mean ± SD

Article Snippet: Human MPCs were trypsinized and incubated for 30 min with biotinylated anti-human PDGFRα (Cat# BAF322, R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% FCS, 2 mM EDTA or with control isotypes IgG1 PE (Cat# A07796, Beckman Coulter) and biotinylated goat IgG (Cat# BAF108, R&D Systems).

Techniques: Expressing, Muscles, Isolation

Journal: Journal of Biomedical Science

Article Title: Many but not all pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification after spinal cord injury

doi: 10.1186/s12929-026-01237-y

Figure Lengend Snippet: OSM and IL-1 neutralization in monocyte-conditioned media strongly inhibit hFAPs mineralization. A OSM, IL-1α and IL-1β concentrations quantified in conditioned media from human CD14 + macrophages stimulated with 200 ng/ml Pam2CSK4 (CM Pam2CSK4 ), 200 ng/ml Pam3CSK4 (CM Pam3CSK4 ) or non-stimulated (CM ∅ ). B OSM, IL-1α and IL-1β concentrations in CM Pam2CSK4 , CM Pam3CSK4 and CM ∅ were correlated with hFAP calcium mineralization measured by Alizarin Red staining after 2 weeks of culture in osteogenic conditions in the presence of the conditioned media. Each dot represents a different conditioned medium sample. C–F Mouse anti-human OSM antibody, isotype control antibody and IL-1RA were used to neutralize OSM and IL-1 in human CD14 + monocyte conditioned media as indicated below each chart. Calcium mineralization of hFAPs cultured for 2 weeks in osteogenic conditions with C CM Pam2CSK4 or D CM Pam3CSK4 was measured using Alizarin Red staining and quantified by spectrophotometry. E, F RUNX2 protein quantification by Western blot using Stain-Free normalization for hFAPs cultured for 2 weeks in osteogenic conditions with E CM Pam2CSK4 or F CM Pam3CSK4 . In C-F, each dot represents a hFAP individual donor. Bars represent mean ± SD, One-way ANOVA Dunnett’s multiple comparison test versus CM ∅ in ( A ) and ( B ), versus CM Pam2CSK4 in ( C ) and ( E ), or versus CM Pam3CSK4 in ( D ) and ( F ), * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human MPCs were trypsinized and incubated for 30 min with biotinylated anti-human PDGFRα (Cat# BAF322, R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% FCS, 2 mM EDTA or with control isotypes IgG1 PE (Cat# A07796, Beckman Coulter) and biotinylated goat IgG (Cat# BAF108, R&D Systems).

Techniques: Neutralization, Staining, Control, Cell Culture, Spectrophotometry, Western Blot, Comparison

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Cdc6 protein levels are only detected in differentiated endoreplicating megakaryoblastic cells. (A) Exponentially growing (−) and TPA-treated (+) HEL, HA1 (Dmescargot-expressing HEL-derived line), K562 and KEB (cyclin E-overexpressing K562-derived line) cells were collected, and whole protein extracts were obtained. Total protein, 30 μg, was subjected to SDS-PAGE and detected by Western blotting with anti-cdc6, -cdt1, -geminin, and -cyclin E antibodies. A portion of total protein staining of transferred gel is shown as a loading control. Asterisk marks the faster migrating cyclin E bands, which correspond to the protein overexpressed in KEB cells. (B) DNA content pattern of these cells is shown as analyzed by flow cytometry. Vertical axis, relative number of cells; horizontal axis, relative red fluorescence (FL2) in a logarithmic scale, indicating DNA content per cell. The positions of peaks representing cells with a DNA content equal to 2C, 4C, 8C, and 16C are indicated. The ability of these cells to achieve (+), or not (−), polyploid DNA content is indicated.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: Expressing, Derivative Assay, SDS Page, Western Blot, Staining, Flow Cytometry, Fluorescence

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Cdc6 stabilization is concomitant with the establishment of the first endoreplication cycle. Whole protein extracts from HEL (A) and K562 (B) cells were obtained before treatment (0 h) or at 4, 8, 15, or 24 h after differentiation induction. Total protein, 30 μg, was subjected to SDS-PAGE and detected by Western blotting with anti-cdc6, -geminin, and -cyclin E antibodies. A portion of the total protein staining of transferred gel is shown as a loading control. Flow cytometry analysis of HEL and K562 DNA content is shown. (C) An identical experiment performed with extracts obtained form HEL and K562 cells at the indicated times after TPA treatment.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: SDS Page, Western Blot, Staining, Flow Cytometry

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Nuclear cdc6 levels are maintained in endoreplicating megakaryoblastic cells. (A) Exponentially growing (−) and 48-h TPA-treated (+) HEL, HA1, K562, and KEB cells were collected. Protein from nuclear and cytoplasmic fractions was extracted. Nuclear or cytoplasmic total protein, 100 μg, was subjected to SDS-PAGE and detected by Western blotting with anti-cdc6, -cyclin A, and -cyclin E antibodies. Blots were stripped and reincubated with anti-PCNA and −I 75 Bα antibodies to assess the purity of subcellular fractions. (B) Exponentially growing (−) or TPA-treated (+) HEL cells were collected. Cdt1 and geminin expression was detected by Western blot in both nuclear (Nuc) and cytoplasmatic (Cyt) fractions. (C) Nuclear and cytoplasmic protein extracts from exponentially growing (−) or differentiated (+) K562 and KEB cells were analyzed by Western blotting with anti-cdt1 and -geminin antibodies. Portions of the total protein staining of transferred gels are shown as loading controls.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: SDS Page, Western Blot, Expressing, Staining

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Cdc6 is stabilized in K562 cells with high levels of cyclin E. (A) KEB cells were cultured for 48 h in the absence (−) or the presence (+) of TPA and total RNA was extracted. RNA, 20 μg, was analyzed by Northern blot with full-length cdc6 cDNA as a probe. The same blot was incubated with a probe recognizing 28S ribosomic RNA and is shown as a loading control. (B) K562 and KEB cells were transiently transfected with either pMT or pMTcdc6 plasmids. Nontransfected, pMT-transfected, and pMTcdc6-transfected cells were allowed to grow exponentially (−) or treated with TPA for 48 h (+). Whole protein fractions were obtained, and Western blot analysis with anticdc6 antibody was performed. An overexposed film is also shown to stress the limited expression of myc-tagged cdc6. (C) pMTcdc6-transfected KEB cells, 2 × 104, were cytospun, fixed with formaldehyde, permeabilized with TX100, and incubated sequentially with anti-myc 9E10 and Alexa Fluor 488–conjugated anti-mouse Ig antibodies. Nuclei were counterstained with DAPI. The figure shows the maximal projection (blue, green, and merge) of an image at 1024 × 1024 resolution. (D) K562 cells were transiently cotransfected with pMTcdc6 and pcDNA-cycE or the corresponding empty plasmids. Either exponentially growing (−) or TPA-treated (+) cells were analyzed by Western blot using anti-cdc6 and -cyclin E antibodies. (E) Western blot analysis with anti-cdc6 and -cyclin E antibodies of TPA-treated pMT-, pMT-cdc6 wild-type (pMTwt)- or pMT- cdc6 5xA mutant (pMT5xA)-transfected cells, and cells cotransfected with pMTwt or pMT5xA plasmids, together with pCDNA-cyclin E are shown. Portions of the total protein staining of transferred gels are shown as loading controls. Both D and E show overexposed blots.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: Cell Culture, Northern Blot, Incubation, Transfection, Western Blot, Expressing, Mutagenesis, Staining

Journal: Cell Death & Disease

Article Title: The novel c-Met inhibitor cabozantinib overcomes gemcitabine resistance and stem cell signaling in pancreatic cancer

doi: 10.1038/cddis.2013.158

Figure Lengend Snippet: Long-term treatment with cabozantinib changes the level of anti- and pro-apoptotic proteins. ( a ) Proteins were isolated from parental BxPc-3 cells and the derived subclone XL-7. Binding of proteins to antibodies spotted in duplicate to the membrane of a Human Apoptosis Array was detected using biotinylated secondary antibodies, streptavidin-HRP and chemiluminescence. The pixel density was quantified using ImageJ software and normalized to the mean pixel intensity of the reference spots A1, A12 and E1 on the membrane. Spot D12 is the PBS-negative control. ( b ) mRNA from parental BxPc-3 cells and derived subclones XL-1 to XL-7 was harvested and expression of Bcl-2 and Survivin were examined by qRT-PCR. ( c ) Likewise, expression of anti-apoptotic (Bcl-2, Survivin, XIAP, cIAP2) and pro-apoptotic (p53, Bim) proteins were examined by western blot analysis

Article Snippet: The following antibodies were used: Mouse mAb against human XIAP (BD Biosciences, San Jose, CA, USA), rabbit mAb against human c-Met, SOX2, p53 (Cell Signaling, Danvers, MA, USA), Ki67 (Epitomics, Burlingame, CA, USA), rabbit pAb against human Bcl-2 (Cell Signaling), Bim (Abcam, Cambridge, UK), cIAP2 (Epitomics), survivin and p-Met (R&D Systems, Minneapolis, MN, USA).

Techniques: Isolation, Derivative Assay, Binding Assay, Software, Negative Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell reports. Medicine

Article Title: Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

doi: 10.1016/j.xcrm.2023.101134

Figure Lengend Snippet: Figure 3. SARS-CoV-2 IgG and neutralizing antibody responses (A and B) Serum IgG titers against wild-type SARS- CoV-2 RBD protein (A) and pseudovirus neutralizing antibody titers (ID50) against SARS-CoV-2 variants (B) for non-elderly (left side) and elderly (right side) participants (n = 10 per dose group, and n = 6 for the placebo group). Participants received one injection of VLPCOV-01 (0.3, 1.0, or 3.0 mg), 30 mg BNT162b2, or placebo on day 1 (week 0). Logarithmic values are reported as geometric mean titers for serum IgG and neutralizing antibody against pseudovirus. Bars indicate 95% CIs. (C) The correlation between serum neutralizing antibody titers against pseudovirus Wuhan (wild type) and IgG antibody titers against SARS-CoV-2 RBD protein following booster vaccination. Pearson’s product-moment correlation coefficient and p value were calculated following log trans- formation of source data (r = 0.950, p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human CD154-FITC (clone: TRAP1) BD Biosciences cat# 555699; RRID: AB_396049 Mouse anti-human CD3-BUV615 (clone: SP34-2) BD Biosciences cat# 751249; RRID: AB_2875266 Mouse anti-human CD4-PE-Cy5.5 (clone: S3.5) Thermo Ficher Scientific cat# MHCD0418; RRID: AB_10376013 Mouse anti-human CD8-BUV563 (clone: RPA-T8) BD Biosciences cat# 612914; RRID: AB_2870200 Mouse anti-human CD27-PE-Cy5 (clone: 1A4CD27) Beckman coulter cat# 6607107 Mouse anti-human CD45RO-BUV805 (clone: UCHL1) BD Biosciences cat# 748367; RRID: AB_2872786 Mouse anti-human IFN-g-BV786 (clone: 4S.B3) BioLegend cat# 502542; RRID: AB_2563882 Mouse anti-human TNF-BV650 (clone: MAb11) BioLegend cat# 502938; RRID: AB_2562741 Rat anti-human IL-13-BV421 (clone: JES10-5A2) BD Biosciences cat# 563580; RRID: AB_2738290 Mouse anti-human IL-21-Ax647 (clone: 3A3-N21) BD Biosciences cat# 560493; RRID: AB_1645421 Mouse anti-human IL-4-PE-Cy7 (clone:8D4-8) BD Biosciences cat# 560672; RRID: AB_1727547 Mouse anti-human IL-17A-BV605 (clone: BL168) BioLegend cat# 512326; RRID: AB_2563887 Rat anti-human IL-2-BUV737 (clone: MQ117H12) BD Biosciences cat# 612836 Mouse anti-human CD107A-BV711 (clone: H4A3) BioLegend cat# 328640; RRID: AB_2565840 Mouse anti-human MIP1b-Alexa700 (clone: D21-1351) BD Biosciences cat# 561278; RRID: AB_10612008 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1 (AS35) ACROBiosystems cat# SAD-S35 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG2 (AS35) ACROBiosystems cat# SAD-S66 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG3 (AS35) ACROBiosystems cat# SAD-S67 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG4 (AS35) ACROBiosystems cat# SAD-S68 Mouse anti-Human IgG1 Fc Secondary Antibody, HRP Thermo Fisher Scientific cat# MH1715; RRID: AB_2539710 Mouse Anti-Human IgG2 Fc-BIOT HP6002 SouthernBiotech cat# 9070-08; RRID: AB_2796638 Mouse Anti-Human IgG3 Hinge-BIOT HP6050 SouthernBiotech cat# 9210-08; RRID: AB_2796700 Mouse Anti-Human IgG4 Fc-BIOT HP6025 SouthernBiotech cat# 9200-08; RRID: AB_2796692 Biological samples Human PBMCs This study This study Chemicals, peptides, and recombinant proteins Benzonase Nuclease, Purity >90% MERCK Millipore cat# 70746 (Continued on next page) Cell Reports Medicine 4, 101134, August 15, 2023 e1

Techniques: Injection

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: (A) Spleen cells from cotton rats were stimulated with Concanavalin A for 72 hours and lymphoblasts purified by centrifugation on a ficoll-gradient. Subsequently, cells were stained with antisera specific for the human interferon receptor to test for crossreactive binding to cotton rat cells and analyzed by flow cytometry. Antibody specific for human interferon alpha receptor-1 (IFNAR-1) did not react with cotton rat cells (left) whereas antibody specific for human interferon alpha receptor-2 (IFNAR-2) did (right). IFNR specific antibody is shown as black line, controls as gray areas. B. IFNAR-2 antibody was added to cotton rat spleen cells. One hour later different concentrations of a combination of ODN2216 and poly I:C was added. 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments. Significant differences were seen between cells treated with the combination of ODN2216 and poly I:C alone, or the combination of ODN2216 and poly I:C with antibody. IFNAR-1 antibody (not reactive with cotton rat cells) had no effect. C. Cotton rat spleen cells were stimulated with ODN 2216 or poly I:C, with or without the addition of recombinant cotton rat interferon alpha. (One unit equals one picogram of type I interferon.) 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments, and significance was compared to cells without type I interferon treatment. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Purification, Centrifugation, Staining, Binding Assay, Flow Cytometry, Recombinant

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: A) The number of MeV-specific B cells was measured from bone marrow cells of MeV-immune cotton rats. The addition of ODN 2216 and poly I:C individually and in combination increased B cell numbers whereas the addition of sera neutralizing cotton rat interferon alpha and IL-6 reduced this stimulation. Each bar graph represents the mean ± SD of triplicate wells. B) Cotton rats were immunized intranasally (upper panel) or subcutaneously (lower panel) with MeV, or MeV with ODN 2216 and/or pI:C in the presence or absence of human MeV-specific IgG (neutralization titer of 100) which had been injected intraperitoneally one day before immunization. Sera were collected at seven weeks post vaccination and the titer of neutralizing antibody was determined by neutralization assay. Each bar graph represents the average titer of four animals ± SD. The experiment is representative of three experiments. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Neutralization, Injection