Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: Journal of Translational Medicine

Article Title: Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer?

doi: 10.1186/1479-5876-11-214

Figure Lengend Snippet: Summary of the immunocytochemical analysis of CNHCs with antibodies against CD45, CD31, and CAIX

Article Snippet: The filters were incubated with primary antibodies directed against CAIX (rabbit IgG, NB100-417, Novus Biologicals, Littleton, USA; 3.5 μg/ml ), CD31 (mouse IgG1, M0823, Dako, Glostrup, Denmark; 1 μg/ml) or CD45 (mouse IgG1, M0855, Dako, Glostrup, Denmark; 2.4 μg/ml) diluted in Antibody Diluent (Dako, Glostrup, Denmark) for 30 min at RT followed by application of primary antibody enhancer (Dako, Glostrup, Denmark) for 15 min.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer?

doi: 10.1186/1479-5876-11-214

Figure Lengend Snippet: Immunocytochemical analysis of CNHCs with antibodies against the RCC marker CAIX. Clusters of CNHCs cytomorphologically classified as uncertain malignant (−UMF) with cytoplasmic positive staining with antibodies against the RCC marker CAIX (A) . Clusters of CNHC-UMF and -BF without reactivity for CAIX antibodies ( B and C , respectively). A single CNHC-MF with positive cytoplasmic (D) and without staining for CAIX (E) . Single CAIX-negative CNHC-UMF and -BF ( F and G , respectively).

Article Snippet: The filters were incubated with primary antibodies directed against CAIX (rabbit IgG, NB100-417, Novus Biologicals, Littleton, USA; 3.5 μg/ml ), CD31 (mouse IgG1, M0823, Dako, Glostrup, Denmark; 1 μg/ml) or CD45 (mouse IgG1, M0855, Dako, Glostrup, Denmark; 2.4 μg/ml) diluted in Antibody Diluent (Dako, Glostrup, Denmark) for 30 min at RT followed by application of primary antibody enhancer (Dako, Glostrup, Denmark) for 15 min.

Techniques: Marker, Staining

Journal: Nucleic Acids Research

Article Title: p53 coordinates base excision repair to prevent genomic instability

doi: 10.1093/nar/gkw015

Figure Lengend Snippet: Persistent SSBs decrease APE1 levels by affecting its transcription. ( A ) Western blotting analysis on a representative XRCC1 knockdown time-course. TIG-1 cells were incubated with either a control siRNA (72 h), or a XRCC1-targeting siRNA, and harvested at the indicated time points. DNA damage accumulation is highlighted by PAR formation; APE1 is downregulated in a time-dependent manner. Actin was used as loading control. ( B ) Alkaline Comet assay on TIG-1 cells harvested 72 h after XRCC1 depletion shows accumulation of SSBs ( N = 9). ( C ) Histogram showing the downregulation in APE1 protein amount 72 h after XRCC1 knockdown, as measured by western blotting ( N = 10). ( D ) Histogram showing the downregulation in APE1 transcript level 72 h after XRCC1 depletion, as measured by qPCR ( N = 7). Results depicted in histograms are presented as mean ± SD of the indicated number ( N ) of independent experiments.

Article Snippet: Membranes were probed with the following antibodies: APE1 (Novus Biologicals, NB100–101), XRCC1 (Thermo Scientific, MS-1393-P0), α-actin (Abcam, ab6276), p21 (Cell Signaling Technology, 2947), p53 (Santa Cruz, sc-126), PAR (Trevigen, 4335-AMC-050), histone H2A.X phosphorylated at Ser139 (γH2AX, 05–636, Millipore), Sp1 (Millipore, 07–645), histone H3 (Cell Signaling Technology, 4499).

Techniques: Western Blot, Knockdown, Incubation, Control, Alkaline Single Cell Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: p53 coordinates base excision repair to prevent genomic instability

doi: 10.1093/nar/gkw015

Figure Lengend Snippet: Downregulation of APE1 in response to BER unbalance is dependent of p53. ( A ) Representative western blotting analysis on TIG-1 cells depleted of XRCC1 and p53. APE1 is downregulated upon XRCC1 knockdown in a p53-dependent manner. Actin was used as loading control. ( B ) Left: histogram showing the quantification of APE1 protein amount in the experiment showed in panel A ( N = 5). Right: histogram illustrating the amount of APE1 transcript upon combined XRCC1/p53 depletion, as measured through qPCR ( N = 3). Results are expressed as mean ± SD of the indicated number ( N ) of independent experiments. ( C ) Boxplot showing the distribution of APE1 staining intensity (in arbitrary units) in p53 low versus p53 overexpressing cells. The dashed line highlights the median APE1 intensity in p53 low cells ( N > 8000). ( D ) Representative high-throughput immuno-fluorescence pictures showing TIG-1 cells stained for APE1 (right panel) and p53 (middle panel) after transfection with a p53 expressing plasmid. Cells that downregulating APE1 in response to p53 overexpression are marked by a contour line.

Article Snippet: Membranes were probed with the following antibodies: APE1 (Novus Biologicals, NB100–101), XRCC1 (Thermo Scientific, MS-1393-P0), α-actin (Abcam, ab6276), p21 (Cell Signaling Technology, 2947), p53 (Santa Cruz, sc-126), PAR (Trevigen, 4335-AMC-050), histone H2A.X phosphorylated at Ser139 (γH2AX, 05–636, Millipore), Sp1 (Millipore, 07–645), histone H3 (Cell Signaling Technology, 4499).

Techniques: Western Blot, Knockdown, Control, Staining, High Throughput Screening Assay, Fluorescence, Transfection, Expressing, Plasmid Preparation, Over Expression

Journal: Nucleic Acids Research

Article Title: p53 coordinates base excision repair to prevent genomic instability

doi: 10.1093/nar/gkw015

Figure Lengend Snippet: Defective p53 activity leads to failure of the BER coordination system. ( A ) High-throughput microscopy analysis of TIG-1 cells transfected with plasmids expressing wild-type or mutant p53. Boxplots showing the distribution of APE1 staining intensity (in arbitrary units) in p53 low versus p53 overexpressing cells. Each p53 mutant is reported on top of the relevant plot. The dashed line highlights the median APE1 intensity in p53 low cells ( N > 5000). NS: not statistically significant at P < 0.05. ( B ) Representative western blotting analysis comparing WI38 and WI38 (SV40) cells upon transfection with the indicated siRNAs. Failure to downregulate Sp1 correlates with the inability to modulate APE1. Actin was used as loading control. ( C ) qPCR analysis on APE1 transcript in WI38 and WI38 (SV40) cells upon transfection with the indicated siRNAs. APE1 transcription is reduced in WI38 cells only. Note the higher transcript content in WI38 (SV40) cells. ( D ) Representative Western blotting analysis comparing WI38 and WI38 (SV40) cells upon transfection with the indicated siRNAs. Failure to modulate BER correlates with γH2AX staining in WI38 (SV40) cells; γH2AX increases further upon XRCC1 depletion. ( E ) Neutral Comet assay on WI38 and WI38 (SV40) fibroblasts shows accumulation of DSBs upon XRCC1-depletion in transformed cells only. Results are expressed as mean ± SD of three independent experiments.

Article Snippet: Membranes were probed with the following antibodies: APE1 (Novus Biologicals, NB100–101), XRCC1 (Thermo Scientific, MS-1393-P0), α-actin (Abcam, ab6276), p21 (Cell Signaling Technology, 2947), p53 (Santa Cruz, sc-126), PAR (Trevigen, 4335-AMC-050), histone H2A.X phosphorylated at Ser139 (γH2AX, 05–636, Millipore), Sp1 (Millipore, 07–645), histone H3 (Cell Signaling Technology, 4499).

Techniques: Activity Assay, High Throughput Screening Assay, Microscopy, Transfection, Expressing, Mutagenesis, Staining, Western Blot, Control, Neutral Comet Assay, Transformation Assay

Journal: Nucleic Acids Research

Article Title: p53 coordinates base excision repair to prevent genomic instability

doi: 10.1093/nar/gkw015

Figure Lengend Snippet: Excessive AP-endonuclease activity in cells with p53 impairment leads to accumulation of genomic instability. ( A ) Representative western blotting analysis on WI38 (SV40) cells shows rescue of γH2AX staining upon co-depletion of XRCC1 and APE1. ( B ) Neutral Comet assay on WI38 (SV40) fibroblasts shows accumulation of DSBs upon XRCC1-depletion and rescue after co-depletion of XRCC1 and APE1. Results are expressed as mean ± SD of three independent experiments. ( C ) Representative western blotting analysis on WI38 (SV40) cells shows rescue of γH2AX staining after depletion of XRCC1 in presence of APE1 inhibitor III (APE1i III, 5 μM for 24 h), or AR03 (2.5 μM for 24 h). Actin was used as loading control in all western blotting experiments.

Article Snippet: Membranes were probed with the following antibodies: APE1 (Novus Biologicals, NB100–101), XRCC1 (Thermo Scientific, MS-1393-P0), α-actin (Abcam, ab6276), p21 (Cell Signaling Technology, 2947), p53 (Santa Cruz, sc-126), PAR (Trevigen, 4335-AMC-050), histone H2A.X phosphorylated at Ser139 (γH2AX, 05–636, Millipore), Sp1 (Millipore, 07–645), histone H3 (Cell Signaling Technology, 4499).

Techniques: Activity Assay, Western Blot, Staining, Neutral Comet Assay, Control

Journal: Biology

Article Title: N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes

doi: 10.3390/biology8020044

Figure Lengend Snippet: qPCR analysis of PGC1-alpha and SIRT1 expression in C2C12 myotubes cultured with L-lactate. C2C12 myotubes were cultured for 2 ( a ) and 6 h ( b ) with L-lactate at different concentrations (0, 10, 30, and 50 mM). In the time between lactate culture and sample collection (one hour), cells were cultured with DM. Error bars show standard deviations. * p < 0.05 from 0 mM. All the experiments were performed three independent times with three replicates each time.

Article Snippet: Membranes were then probed at 4 °C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PGC1-alpha (Novus Biologicals, Centennial, CO, USA) and GAPDH (Abcam, Cambridge, UK).

Techniques: Expressing, Cell Culture

Journal: Biology

Article Title: N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes

doi: 10.3390/biology8020044

Figure Lengend Snippet: qPCR analysis of PGC1-alpha, Cyt C, and SIRT1 in C2C12 myotubes cultured with lactate and N-acetyl-L-cysteine (Nac). C2C12 myotubes were cultured for 2 h with L-lactate (0 and 50 mM) in the presence or absence of Nac (2 mM). Samples for gene expression analysis were obtained at 0 ( a ) and 3 h ( b ) after the culture with lactate and Nac. In the time between lactate culture and sample collection (0 to 3 h), cells were cultured with DM. Error bars show standard deviations. * p < 0.05 from 0 mM. All the experiments were performed three independent times with three replicates each time.

Article Snippet: Membranes were then probed at 4 °C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PGC1-alpha (Novus Biologicals, Centennial, CO, USA) and GAPDH (Abcam, Cambridge, UK).

Techniques: Cell Culture, Gene Expression

Journal: Biology

Article Title: N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes

doi: 10.3390/biology8020044

Figure Lengend Snippet: qPCR analysis for intermittent and continuous exposure to lactate in C2C12 myotubes and Western blot of PGC1-alpha after continuous or intermittent exposure to lactate. C2C12 myotubes were cultured with different concentration patterns of L-lactate (see ). Samples for qPCR analysis were obtained 0 ( a ) and 3 h ( b ) after the exposure series. Samples for Western blot ( c ) were obtained 3 h after the exposure series (see ). In the time between lactate culture and sample collection (zero or three hours), cells were cultured with DM. Error bars show standard deviations. * p < 0.05 from non-lactate exposure (0 × 0). RNA experiments were performed three independent times with three replicates each time, and protein experiments with three independent samples.

Article Snippet: Membranes were then probed at 4 °C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PGC1-alpha (Novus Biologicals, Centennial, CO, USA) and GAPDH (Abcam, Cambridge, UK).

Techniques: Western Blot, Cell Culture, Concentration Assay