Journal: The American journal of pathology

Article Title: Preclinical and Clinical Evidence of Mycobacterium tuberculosis Persistence in the Hypoxic Niche of Bone Marrow Mesenchymal Stem Cells after Therapy.

doi: 10.1016/j.ajpath.2015.03.028

Figure Lengend Snippet: Figure 4 In vivo pimonidazole labeling of CD271þ bone marrowe mesenchymal stem cells (BM-MSCs) containing post-therapy persistent Mycobacterium tuberculosis (MTB). A: Representative flow cytometry panel showing the pimonidazole labeling of CD271þ BM-MSCs obtained from noneMTB-infected healthy mice. The histogram shows hypoxia-inducible factor (HIF)-1a expression of the CD271þ/pimonidazoleþ cell fraction. BM CD271þ/CD45 cells (1 105) obtained from pimonidazole-injected BALB/c mice (aged 6 to 8 weeks) by immunomagnetic sorting were fixed, and stained against pimonidazole (allophycocyanin), CD271 (phycoerythrin PE), and HIF-1a (fluorescein isothiocyanate) for flow cytometry analysis. B: Representative flow cytometry profile showing the pimonidazole staining (APC) of green fluorescent protein (GFP)-H37Rvþ CD271þ BM-MSCs ob- tained from day 90 of isoniazid (INH)-pyrazinamide (PZA)etreated mice. After the completion of the INH and PZA treatment, BALB/c mice (aged 6 to 8 weeks) were injected with pimonidazole, and BM cells were recovered to isolate CD271þ/CD45 cells by immunomagnetic sorting. For the isotype controls, pimonidazole-injected healthy mouse BM was used. The dashed arrows in A and B highlight that, although 73% of noneMTB-infected cells were positive for pimonidazole (A), most cells were pimonidazole positive in MTB-infected mice (B). n Z 6 mice (A); n Z 3 mice (B); n Z 3 in- dependent experiments (A and B).

Article Snippet: Samples were incubated with primary antibody against CD45 (rat clone 30-F11, 1:100; Biolegend, San Diego, CA), CD271 (rat clone 2E3, ab27007, or mouse clone ME20.4, ab8877; Abcam), HIF-1a (rabbit polyclonal, NB100-449; Novus, Littleton, CO), pimonidazole (mouse clone 4.3.11.3, 1:50; Hypoxyprobe, Inc.), CD44 [clone IM7; allophycocyanin (APC) conjugated; Biolegend], and CD73 (clone TY/11.8; Pacific blue conjugated; Biolegend) for 2 hours in staining buffer (4% bovine serum albumin plus 0.1% saponin) at room temperature.

Techniques: In Vivo, Labeling, Cytometry, Infection, Expressing, Injection, Staining

Journal: The American journal of pathology

Article Title: Preclinical and Clinical Evidence of Mycobacterium tuberculosis Persistence in the Hypoxic Niche of Bone Marrow Mesenchymal Stem Cells after Therapy.

doi: 10.1016/j.ajpath.2015.03.028

Figure Lengend Snippet: Figure 6 Mycobacterium tuberculosis (MTB)-harboring CD271þ bone marrowemesenchymal stem cells (BM-MSCs) of post-therapy pulmonary tuberculosis (PTB) subjects exhibit hypoxic phenotype. A: Histogram showing the high hypoxia-inducible factor (HIF)-1a/low CD146 gene expression of CD271þ BM-MSCs cultured in 1% oxygen for a week. The BM-MSCs were commercially obtained. B: Histogram showing the expression of HIF-1a and CD146 in the CD271þ BM-MSCs obtained from seven post-therapy PTB subjects having confirmed MTB-DNA (MTB-DNA þ subjects group) intra- cellular to CD271þ BM-MSCs,12 and four subjects having no history of PTB, and no detectable presence of MTB-DNA intracellular to CD271þ BM-MSCs13

Article Snippet: Samples were incubated with primary antibody against CD45 (rat clone 30-F11, 1:100; Biolegend, San Diego, CA), CD271 (rat clone 2E3, ab27007, or mouse clone ME20.4, ab8877; Abcam), HIF-1a (rabbit polyclonal, NB100-449; Novus, Littleton, CO), pimonidazole (mouse clone 4.3.11.3, 1:50; Hypoxyprobe, Inc.), CD44 [clone IM7; allophycocyanin (APC) conjugated; Biolegend], and CD73 (clone TY/11.8; Pacific blue conjugated; Biolegend) for 2 hours in staining buffer (4% bovine serum albumin plus 0.1% saponin) at room temperature.

Techniques: Gene Expression, Cell Culture, Expressing

Journal: Chromosoma

Article Title: FancJ (Brip1) loss-of-function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase I in mice.

doi: 10.1007/s00412-015-0549-2

Figure Lengend Snippet: Fig. 7 Western blot analysis of whole testis lysates from Fancj+/+ and FancjGT/GT males reveals specific alterations in DNA MMR proteins and BLM. a–g Quantitation of protein levels for BRCA1, BLM, MLH1, MLH3, MSH4, MSH5, and MUS81 normalized to β-tubulin, showing significant increases in protein levels in FancjGT/GT (gray bars) compared to Fancj+/+ (black bars) testis extracts for all except BRCA1 and MUS81

Article Snippet: Primary-antibody incubation was performed for 12 h at 4 °C at 1:1000 dilution (antibodies used are: MUS81 (NBP1-32054 from Novus Biochemicals), MSH4 (ab95096 from Abcam), MSH5 (LS-C101911 from LsBio), MLH1 (550838 from BD pharmingen), β-Tubulin (T8328 from Sigma),β-Actin (A2228 from Sigma), BLM (a gift from Dr. Raimundo Freire), BRIP1/FANCJ (Novus, NBP1-31883), and BRCA1 (a gift from Dr. Satoshi Namekawa).

Techniques: Western Blot, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Muramyl dipeptide potentiates Staphylococcus aureus lipoteichoic acid-induced nitric oxide production via TLR2/NOD2/PAFR signaling pathways

doi: 10.3389/fimmu.2024.1451315

Figure Lengend Snippet: Enhanced SaLTA-induced NO production through MDP is mediated by TLR2 and CD14/MyD88-, and NOD2-dependent pathway. (A) RAW 264.7 cells (500 μl of 5 × 10 5 cells/ml) were treated with SaLTA (0 or 3 μg/ml) and/or MDP (0 or 10 μg/ml) for 24 h. The cells were then stained with anti-mouse TLR2 conjugated with PE or CD14 conjugated with FITC antibodies. For intracellular NOD2, the cells were permeabilized and stained with anti-mouse NOD2 conjugated with PE. The stained cells were then subjected to flow cytometry. Upper , a representative TLR2, NOD2, and CD14 expression determined by flow cytometric analysis. Values given in each histogram indicate mean fluorescence intensity (MFI) of 10,000 events of live cells in the treatment group. Lower , the expression of TLR2, NOD2, and CD14 is presented as mean of MFI ± S.D. of three replicates for each group. Asterisk (*) indicates a significant difference between the indicated treatment groups at P < 0.05. NT, non-treatment group; IC, isotype control. (B, C) BMMs (200 μl of 5 × 10 5 cells/ml) from wild-type, (B) TLR2-, MyD88- CD14- or (C) NOD2-deficient mice were treated with SaLTA (0 or 3 μg/ml) and/or MDP (0 or 10 μg/ml) for 24 h. Then, nitrite levels in the supernatants were measured. The data represent mean ± S.D. of three replicates for each group. * indicates a significant difference compared to the control group at P < 0.05.

Article Snippet: For flow cytometric analysis, antibodies specific to TLR2 conjugated with phycoerythrin (PE, Catalog No. 12-9021-82), and to CD14 conjugated with fluorescein isothiocyanate (FITC, Catalog No. 123308) were obtained from eBioscience (San Diego, CA, USA) and Biolegend (San Diego, CA, USA), respectively, while antibody specific to NOD2 conjugated with PE (Catalog No. NB100-524PE) was purchased from Novus (Los Angeles, CA, USA).

Techniques: Staining, Flow Cytometry, Expressing, Fluorescence, Control

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of MRE11 phosphorylation kinetics at MRE11S676S678. ( A ) MRE11pS676pS678 antibody immunoprecipitate the phosphorylated form of MRE11 after ionizing radiation (IR). Western blot (MRE11) of immunoprecipitated MRE11 using an antibody against MRE11S676S678 (nonP) site and the corresponding phosphorylated MRE11pS676pS678 (pSpS) sites from control and ATLD2 lymphoblastoid cells treated with 0 or 10 Gy IR. Unbound sample from each immunoprecipitation was run in parallel and also immunoblotted for total MRE11 (GeneTex). ( B ) Dose-dependent increase of MRE11 phosphorylation following IR. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control and A-T lymphoblastoid cells treated with 0, 2, 5 or 10 Gy IR. ATLD2 cells were also run as a negative control (0 and 10 Gy). Western blot for the MRN complex proteins, MRE11, RAD50 and NBS1. ( C ) Increasing MRE11 phosphorylation over 2 h post 5 Gy. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control lymphoblastoid cells either untreated (−) or harvested at 0.6, 2, 4 or 10 h post 5 Gy IR. ATLD2 lymphoblastoid cells were either untreated (−) or harvested at 2 and 10 h post 5 Gy IR and run as a negative control. ( D ) MRE11 phosphorylation in response to DNA double strand break inducing agents. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control cells treated with either IR, camptothecin (CPT), hydrogen peroxide (H2O2), etoposide (ETOP), cisplatin (CISP) or methlymethanesulfonate (MMS).

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of ATM signalling, cell survival and chromosomal aberrations in the cell line expressing non-phosphorylatable MRE11S676AS678A. ( A ) Stable MRN complex in WT and non-phosphorylatable mutant MRE11 corrected cell lines. Western blot of immunoprecipitated MRE11 from control (MCR5) and ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines. Also immunoblotted for NBS1 and RAD50. ( B ) ATM signalling in the non-phosphorylatable mutant MRE11 cell line is comparable to the WT corrected cell line. Total cell extracts from ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines were extracted 30 min post 5 Gy (+) or left as unirradiated controls (−) and western blotted for ATM S1981 autophosphorylation and total ATM, as well as the ATM kinase substrates, SMC1 and KAP1. Tubulin was immunoblotted as loading control. ( C ) The non-phosphorylatable mutant MRE11 cell line displays increased cellular sensitivity to IR. Plot of percent survival after 0, 1, 2, 3, 4 and 5 Gy IR. NFF (control), ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), A-T, ATLDS676A (S676A) and ATLDS678A (S678A) cell lines were treated with increasing doses of IR and percent survival assessed by clonogenic cell survival assay. The mean ± standard deviation is plotted from 3 independent experiments. ( D ) Non-phosphorylatable mutant MRE11 cell line is unable to correct radiation induced chromosomal aberrations. Aberrations (chromatid breaks, chromosome breaks and interchanges) were scored from Giemsa stained metaphases in 2 Gy irradiated NFF (control), A-T, ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), ATLDS676A (S676A) and ATLDS678A (S678A) cell lines. Induced chromosomal aberrations (ICA) were totalled and divided by number of metaphases as indicated in parentheses.

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Expressing, Mutagenesis, Western Blot, Immunoprecipitation, Control, Clonogenic Cell Survival Assay, Standard Deviation, Staining, Irradiation