Journal: Infection and Immunity

Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

doi: 10.1128/IAI.00044-19

Figure Lengend Snippet: Chemical genetics screen identifies pathways that inhibit intracellular growth of B. neotomae. (A) Inhibitory effects of selected small molecules on intracellular replication in J774A.1 host cells at 48 h postinfection. Both B. neotomae (Bn) luminescence and CFU were measured in parallel. Data shown are means ± SD from at least two independent experiments, 3 replicates for luminescence data and 2 replicates for CFU data. Concentrations of the following tested compounds were in general above the IC50 (Table 1) and substantially below the CC50: arcyriaflavin A (3.6 μM), BX912 (3.6 μM), IKK16 (3.2 μM), I3M (3.6 μM), Ki20227 (2.0 μM), CYT387 (1.4 μM), Rhodblock6 (6.9 μM), SP600125 (10.2 μM), esomeprazole (10.2 μM), NS8593 (4.7 μM), and pinacidil (10.2 μM). (B) Inhibitory effects of selected small molecules on intracellular replication in primary murine bone marrow-derived macrophages (BMDM) at 48 h postinfection based on luminescence signals. Data shown are means ± SD from 4 replicates. Saponin (0.2%) and 0.3% DMSO in the absence of infection were used as positive and negative cytotoxicity controls, respectively. Significant suppression of luminescence was observed for all inhibitors in comparison with the DMSO control (P < 0.0001 by ANOVA and Dunnett’s post hoc multiple-comparison tests). (C) Effect of kinase target siRNA on intracellular growth of B. neotomae in J774A.1 host cells as assessed by luminescence. Data points represent means ± SD from at least three independent experiments. Significant suppression of luminescence (*, P < 0.001) was observed for siRNA knockdowns compared to the NTsi control. (D) Effect of siRNA knockdowns on mRNA expression assayed using same experimental protocol as the one described for panel C. Data points represent means ± SD from three independent samples and were normalized to the β-actin level within each sample and then to expression in untreated controls.

Article Snippet: NS8593 , Alomone Labs , N-196.

Techniques: Derivative Assay, Infection, Expressing

Journal: Infection and Immunity

Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

doi: 10.1128/IAI.00044-19

Figure Lengend Snippet: Compound potency, cytotoxicity, and selectivity

Article Snippet: NS8593 , Alomone Labs , N-196.

Techniques:

Journal: Infection and Immunity

Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

doi: 10.1128/IAI.00044-19

Figure Lengend Snippet: Strains and key resources used in this study a

Article Snippet: NS8593 , Alomone Labs , N-196.

Techniques: Bicinchoninic Acid Protein Assay, Transfection, Software

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation of [Ca 2+ ] i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

doi: 10.1186/s12958-020-00643-7

Figure Lengend Snippet: Effect of TRPM7 inhibitor NS-8593 on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution

Article Snippet: The details of Ca 2+ channel blockers used in the study are described below: Thapsigargin (10,522, Cayman, Michigan, USA), a SERCAs inhibitor [ ] stored in DMSO in 10 mM, with gradient working concentrations of 0.5, 1, and 10 μM; NS-8593 (N-195, Alomone, Jerusalem BioPark, Jerusalem, Israel), a TRPM7 inhibitor [ ], stored in DMSO in 10 mM, with working gradient concentrations of 0.1, 1, and 5 μM; Mibefradil (Mib, HY-15553, MCE, NJ 08852, USA), a T-type Ca 2+ channels blocker [ ], stored in water in 10 mM, with working gradient concentrations of 0.5, 5, and 10 μM; GSK-7975A (HY12507, MCE, NJ 08852, USA), an Orai1blocker [ ], stored in DMSO in 10 mM, with gradient concentrations of 10, 100 μM, and 1 mM.

Techniques: Activation Assay, Fluorescence, Inhibition

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation of [Ca 2+ ] i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

doi: 10.1186/s12958-020-00643-7

Figure Lengend Snippet: Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p < 0.05 (*) was considered statistically significant. (X) indicates data unavailable

Article Snippet: The details of Ca 2+ channel blockers used in the study are described below: Thapsigargin (10,522, Cayman, Michigan, USA), a SERCAs inhibitor [ ] stored in DMSO in 10 mM, with gradient working concentrations of 0.5, 1, and 10 μM; NS-8593 (N-195, Alomone, Jerusalem BioPark, Jerusalem, Israel), a TRPM7 inhibitor [ ], stored in DMSO in 10 mM, with working gradient concentrations of 0.1, 1, and 5 μM; Mibefradil (Mib, HY-15553, MCE, NJ 08852, USA), a T-type Ca 2+ channels blocker [ ], stored in water in 10 mM, with working gradient concentrations of 0.5, 5, and 10 μM; GSK-7975A (HY12507, MCE, NJ 08852, USA), an Orai1blocker [ ], stored in DMSO in 10 mM, with gradient concentrations of 10, 100 μM, and 1 mM.

Techniques:

Journal: Scientific Reports

Article Title: TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

doi: 10.1038/srep34236

Figure Lengend Snippet: ( a,f ) Whole-cell voltage clamp recordings from oocytes ( a–c ) and eggs ( d–f ). ( a ) Whole-cell patch clamp recording in response to 80 μM Naltriben (red bar). ( b ) Averaged current recorded at +80 mV (7 ± 2, 19 pA/pF for recordings in basal conditions (No external Mg 2+ , red symbol); n = 4. 9, 4 ± 2, 4 pA/pF for current responses to 80 μM Naltriben; n = 4). ( c ) Current evoked from a voltage ramp from −100 to +100 mV in response to 30 μM NS8593 (cyan). Insert: Averaged current recorded at +80 mV (9 ± 4, 3 pA/pF for basal responses; n = 5. 3, 7 ± 1, 7 pA/pF for responses to 30 μM NS8593; n = 5). ( d ) Representative trace of a voltage-clamp recording on an egg in response to 80 μM Naltriben (red bar). ( e ) Averaged current recorded at +80 mV (4, 9 ± 1, 8 pA/pF for recordings in basal conditions (No external Mg 2+ , red symbol); n = 3. 7, 9 ± 2, 1 pA/pF for current responses to 80 μM Naltriben; n = 3). ( f ) Current-Voltage (I-V) relation in response to a voltage ramp (−100 mV to +100 mV) in basal conditions (no external Mg 2+ , red trace) and in response to 30 μM NS8593 (cyan). Insert: Averaged current recorded at +80 mV (5, 4 ± 1, 5 pA/pF for basal responses; n = 6. 1, 6 ± 0, 9 pA/pF for responses to 30 μM NS8593; n = 6). ± SD. ***P < 0.001, *P < 0.05.

Article Snippet: NS8593 (SIGMA), Naltriben (SIGMA).

Techniques: Patch Clamp

Journal: Scientific Reports

Article Title: TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

doi: 10.1038/srep34236

Figure Lengend Snippet: Spontaneous [Ca 2+ ] i oscillations were induced and measured at RT in response to increasing concentrations of extracellular Ca 2+ and 10 mM Sr 2+ in WT oocytes. ( a ) Left panel , [Ca 2+ ] i oscillations were evaluated in response to increased extracellular Ca 2+ (black horizontal bars). Figure shows representative traces for 3 oocytes. n = 9. Right panel : Oocytes were exposed to 10 μM NS8593 (cyan bar) and Ca 2+ oscillations were evaluated after increasing external Ca 2+ concentration from 2 or 5 mM (black horizontal bars). n = 13. ( b ) Left panel : Representative traces for 3 oocytes showing Sr 2+ -induced oscillations. n = 5, oocytes that show 3 or more oscillations. Right panel : Sr 2+ -induced oscillations were blocked by exposure to 10 μM NS8593 (cyan bar). n = 9. ( c ) Oscillations induced by Sr 2+ at 33–35 °C ( left panel , n = 5. Figure shows representative traces for 3 eggs) are not blocked by 10 μM NS8593 (cyan bar; right panel, n = 7. Figure shows representative traces for 3 eggs).

Article Snippet: NS8593 (SIGMA), Naltriben (SIGMA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

doi: 10.1038/srep34236

Figure Lengend Snippet: ( a ) Whole-cell patch clamp recordings in blastomeres from 2-cell stage obtained after fertilization. Averaged current at +80 mV is showed. Red symbols: Basal current, 9, 7 ± 3, 3 pA/pF, n = 4. Cyan: Current in response to 30 μM NS8593, 3, 2 ± 0, 6 pA/pF, n = 4. ( b ) Comparison of basal current density between GV oocytes (orange symbols; 8, 1 ± 3, 5 pA/pF, n = 9), MII eggs (blue diamonds; 5, 2 ± 1, 5 pA/pF, n = 9) and 2-cell stage blastomers (violet symbols; 9, 7 ± 3, 3 pA/pF, n = 4) in absence of external Mg 2+ measured at +80 mV. ± SD. *P < 0.05. Scale bar: 50 μm.

Article Snippet: NS8593 (SIGMA), Naltriben (SIGMA).

Techniques: Patch Clamp

Journal: Scientific Reports

Article Title: TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

doi: 10.1038/srep34236

Figure Lengend Snippet: NS8593 inhibits pre-implantation embryo development to the blastocyst stage of mouse WT CD1 zygotes.

Article Snippet: NS8593 (SIGMA), Naltriben (SIGMA).

Techniques:

Journal: Scientific Reports

Article Title: TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

doi: 10.1038/srep34236

Figure Lengend Snippet: NS8593 delays developmental progression of mouse WT CD1 zygotes prior to the morula stage.

Article Snippet: NS8593 (SIGMA), Naltriben (SIGMA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts

doi: 10.3390/ijms22083992

Figure Lengend Snippet: Whole-cell patch-clamp recordings of TRPM7 currents in HAT-7 cells. ( A ) TRPM7-related currents at different intracellular Mg 2+ concentrations ( n = 14). Effects of inhibitors on TRPM7 currents induced by a nominally Mg 2+ -free intracellular solution: ( B ) NS8593 (20 µM, n = 5) and ( C ) FTY720 (2 µM, n = 5). Effects of TRPM7 activators: ( D ) currents evoked by mibefradil (50 µM) in the presence of 0.9 mM ( n = 5) and 3.6 mM [Mg 2+ ] i ( n = 4); ( E ) currents evoked by naltriben (50 µM) in the absence and presence of NS8593 (50 µM, n = 4) at physiologic [Mg 2+ ]. Data are presented as means ± SEM.

Article Snippet: Following activators and inhibitors were used in functional studies: naltriben (Tocris Bioscience, Bristol, UK), mibefradil (Tocris Bioscience, Bristol, UK), NS8593 (Tocris Bioscience, Bristol, UK), thapsigargin (Sigma Aldrich, St. Louis, MO, USA), FTY720 (Sigma Aldrich, St. Louis, MO, USA) and BTP2 (Merck KGaA, Dramstadt, Germany).

Techniques: Patch Clamp

Journal: International Journal of Molecular Sciences

Article Title: TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts

doi: 10.3390/ijms22083992

Figure Lengend Snippet: Changes in intracellular Ca 2+ in response to TRPM7-specific activators and inhibitors. ( A , B ) Changes in [Ca 2+ ] i evoked by naltriben (100 µM, applied at 120 s) in the presence ( n = 9) and absence of extracellular Ca 2+ , and in the presence of 20 µM NS8593 (applied at 0 s, n = 5). ( C , D ) Changes in [Ca 2+ ] i evoked by mibefradil (50 µM, applied at 120 s) in the presence ( n = 12) and absence ( n = 6) of extracellular Ca 2+ , and following pretreatment with 100 nM thapsigargin ( n = 4). Ca 2+ was present in the bath solution unless the legend states otherwise. Data are presented as representative traces of changes in fura-2 fluorescence ratio normalized to baseline, and as mean peak values ± SEM (** p < 0.01, *** p < 0.001).

Article Snippet: Following activators and inhibitors were used in functional studies: naltriben (Tocris Bioscience, Bristol, UK), mibefradil (Tocris Bioscience, Bristol, UK), NS8593 (Tocris Bioscience, Bristol, UK), thapsigargin (Sigma Aldrich, St. Louis, MO, USA), FTY720 (Sigma Aldrich, St. Louis, MO, USA) and BTP2 (Merck KGaA, Dramstadt, Germany).

Techniques: Fluorescence