Journal: Science Advances

Article Title: Human DNA topoisomerase I poisoning causes R loop–mediated genome instability attenuated by transcription factor IIS

doi: 10.1126/sciadv.adm8196

Figure Lengend Snippet: ( A and B ) Enrichment analysis of observed R loop gain with and without (w/o) DSBs at early, early/mid, and late IZs (A) and at early, early/mid, and late IZs and in TAD classes (B) assessed as in . ( C ) PLA assay of PCNA with RNAPII in cells expressing (+ doxy) or not (− doxy) TFIISm. CTRL, control samples using the indicated antibody only. Fold changes of CPT-treated versus untreated and induced versus uninduced samples are reported over each bar. Mean of analyzed cells is 800. Statistical significance was calculated with one-tailed paired t test. ( D ) Experimental protocol of 1-hour CPT treatment with single (top) or dual (bottom) labeling of HeLa cells. ( E ) Representative images of the possible cases of dual labeled cells. ( F to H ) Levels of micronuclei triggered in late G 1 , late S, or early/mid S phase [as in (E)], respectively. Micronucleus levels are reported as micronuclei (MNi) per 100 cells (left) or as fold change in RNase H1–expressing (RH) versus control cells (right). Means of analyzed cells are 250 (F), 150 (G), and 400 (H). Statistical significance was calculated comparing micronucleus distribution of treated over untreated samples with two-tailed Mann-Whitney test. ( I and J ) γH2AX detected by immunofluorescence microscopy at the end of EdU administration (I) or at the end of BrdU administration (J) as in (D). Levels of nuclear fluorescence are reported as fold change over untreated cells (number of analyzed cells is 450). Statistical significance was calculated considering treated over untreated samples with one-tailed ratio paired t test. In (C) and (F) to (J), bars show means ± SEM of three biological replicates. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human cancer HCT116 cells, U2OS, and HeLa cell lines were purchased from American Type Culture Collection (LGC Standards S.r.l., Milan, Italy).

Techniques: Expressing, Control, One-tailed Test, Labeling, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Microscopy, Fluorescence

Journal: Proteome Science

Article Title: An antibody microarray analysis of serum cytokines in neurodegenerative Parkinsonian syndromes

doi: 10.1186/1477-5956-10-71

Figure Lengend Snippet: Validation of the serum cytokine array experiment in the initial screening cohort

Article Snippet: ELISA kits specific for human MCP-4 (DY327) and prolactin (DY682) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: Osteoarthritis and cartilage

Article Title: Role of the C-C chemokine Receptor-2 in a Murine Model of Injury-induced Osteoarthritis

doi: 10.1016/j.joca.2016.11.004

Figure Lengend Snippet: Levels of (A) CCL12 and (B) other’s CCR2 ligands (CCL2, CCL8, CCL7, CCL13) were measured by immunohistochemistry (IHC) in articular cartilage sections obtained from medial knee joints of mice at the indicated time points after DMM or sham control surgery. Brown is immunopositive staining. DMM-operated knees at 4, 8 and 12 weeks post-surgery were immunostained with antibodies to CCR2 and protein levels where visualized in (C) medial tibial cartilage, (D) chondrocytes of the growth plate and (E) synovium. Images are representative of 6 different mice for each of the experimental points described. Scale bars are 100 µm.

Article Snippet: Rabbit polyclonal anti-CCL2 (orb13563), CCL8 (orb13565), CCL7 (orb13567), CCL13 (orb13285) and CCL12 (orb13284) antibodies were from Biorbyt; rabbit polyclonal anti-CCR2 was from Novusbio (NB100-701); anti-CollagenX (ab58632), anti-Osteocalcin (ab10911) and mouse monoclonal anti-MMP-13 (ab3208) antibodies were from Abcam; The CCR-2 antagonist RS-504393 was from Tocris Bioscience.

Techniques: Immunohistochemistry, Staining

Journal: Osteoarthritis and cartilage

Article Title: Role of the C-C chemokine Receptor-2 in a Murine Model of Injury-induced Osteoarthritis

doi: 10.1016/j.joca.2016.11.004

Figure Lengend Snippet: Protein levels of CCL12 were visualized by IHC in the bone tissues (A) 2 wks after DMM/sham, as well as (B) 4 and 8 wks post-surgery. Protein levels of (C) CCL2, CCL8, CCL7, CCL13, as well as (D) CCR2 were visualized in bone tissue by IHC at the indicated time points. (E) Protein levels and mRNA expression of Col10 were visualized by IHC and ISH, respectively, in medial femurs obtained from mice 1wk after DMM/sham surgery. Osteophytes were visualized by (F) μCT analyses and (G) OCN protein/mRNA levels (IHC and ISH) in medial femurs and tibiae obtained from mice 2wks after DMM/sham surgery. Images are representative of 6 different mice for each of the experimental points described. Scale bars are 100 µm.

Article Snippet: Rabbit polyclonal anti-CCL2 (orb13563), CCL8 (orb13565), CCL7 (orb13567), CCL13 (orb13285) and CCL12 (orb13284) antibodies were from Biorbyt; rabbit polyclonal anti-CCR2 was from Novusbio (NB100-701); anti-CollagenX (ab58632), anti-Osteocalcin (ab10911) and mouse monoclonal anti-MMP-13 (ab3208) antibodies were from Abcam; The CCR-2 antagonist RS-504393 was from Tocris Bioscience.

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Adaptively evolved human oral actinomyces‐sourced defensins show therapeutic potential

doi: 10.15252/emmm.202114499

Figure Lengend Snippet: An inhibition‐zone‐based assay for evaluating the stability of AMSIN in H 2 O or normal mouse serum. The bacterium used was BM and the peptide dose was 0.2 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann–Whitney U ‐test (* P < 0.05, ns: no significance; exact P values are listed in Table ). Hemolysis by AMSIN. Meucin‐18 was used as a positive control. Mean ± SD of three technical replicates is displayed. Comparison of cytotoxic effects of AMSIN and LL‐37 on HL‐60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk; * P < 0.05, *** P < 0.001, ns: no significance; exact P values are listed in Table ). An inhibition‐zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann–Whitney U ‐test (ns: no significance; exact P values are listed in Table ). The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady‐state current traces after administering 100 μM AMSIN. Peptide‐induced IL‐8 release. A549 cells were treated with AMSIN or LL‐37 for 24 h. IL‐8 levels in culture supernatants from the FBS‐containing medium ( left ) or the serum‐free medium ( right ) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk or ns; * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significance; exact P values are listed in Table ). Peptide‐induced MCP‐1 release. HL‐60 cells were treated with AMSIN or LL‐37 for 24 h. MCP‐1 levels in culture supernatants from the FBS‐containing medium ( left ) or the serum‐free medium ( right ) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk or ns; * P < 0.05, ** P < 0.01, ns: no significance; exact P values are listed in Table ). Surface plasmon resonance (SPR)‐based assay detecting anti‐AMSIN antibodies in mouse serum using Biacore T100. Sera were taken from three AMSIN‐treated mice (Samples 1–3) and one control mouse only injected with 0.9% NaCl. The sensorgrams present data of subtracting the background SPR signals from the control. Note: the abnormal peaks at 30 and 90 s yielded by the equipment due to sample injections were artificially removed. Insect, schematic diagram of SPR experiment in which 1:100 diluted serum was used as analyte to flow over the AMSIN‐immobilized CM5 chip. Treatment of pneumococcal pneumonia. Five mice per sampling point were inoculated with SP D39 through the nasopharynx and 24 h later the animals received AMSIN or penicillin (as a positive control) at different doses (AMSIN, at the doses of 8.35, 16.7, and 33 mg per kg; penicillin at 8.35, 16.7, and 30 mg per kg) via intramuscular injection (i.m.). One day after treatment, the animals were necropsied and their lungs were removed and homogenized for the determination of the level of SP , as described in the Materials and Methods section. Each point represents the determination from a single animal and the line shows the mean log value. Mean ± SD of five to ten biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by red asterisks; ** P < 0.01, *** P < 0.001 denotes significant reduction in CFU compared with the saline group; exact P values are listed in Table ). Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle‐treated control group was zero out of 11 mice and the treatment group contained six mice and all survived. Source data are available online for this figure.

Article Snippet: Medium was aspirated and replaced with fresh complete DMEM supplemented with 10% FBS or serum‐free medium, both containing different concentrations of polypeptides for 24 h. Then, the supernatants were harvested for quantification of the IL‐8 amount in A549 by the Human CXCL8/IL‐8 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) and the MCP‐1 amount in HL‐60 by the Human MCP‐1 ELISA Kit (Boster Biological Technology Co., Ltd, Wuhan, China) as per the manufactures’ instructions.

Techniques: Inhibition, MANN-WHITNEY, Positive Control, Comparison, Enzyme-linked Immunosorbent Assay, SPR Assay, Control, Injection, Sampling, Saline, Infection