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. 2015 Sep 28;1:15035. doi: 10.1038/cddiscovery.2015.35

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Copyright © 2015 Cell Death Differentiation Association

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Atg16L1, ROS and ERK regulate Cx43 in the presence of VacA. (a) Control (NC) or Atg16L1 siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43, LC3-II generation and Atg16L1 in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (b) The indicated siRNA-transfected cells were treated with toxin as shown above. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP, Atg16L1 and GAPDH in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from two experiments. Experiments were repeated two times with similar results. (c and d) AZ-521 cells were pretreated with or without 10 mM NAC (left panel) or 10 mM GSH (right panel) for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation in AZ-521 cells was performed by densitometry. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05 and **P<0.02. Experiments were repeated three times with similar results. (e) AZ-521 cells were pretreated with control (DMSO) or 10 mM NAC and then 120 nM heat-inactivated (iV) or wild-type VacA (V) for the indicated times. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. (f) AZ-521 cells were pretreated with 10 μM U0126 for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results. (g) Control (NC) or ERK siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation and ERK levels in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (h) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 4 h and total RNA was extracted as described in Materials and Methods. Cx43 mRNA was measured by real-time qPCR. Data are shown as mean±S.D. of values from two experiments. Results are shown as fold increase of GAPDH as an internal control. Experiments were repeated two times with similar results. (i) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and then reacted with anti-Cx43 antibodies (red) and incubated with DAPI (cyan). Bars represent 20 μm. Experiments were repeated three times with similar results. (j) The indicated siRNA-transfected AZ-521 cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP and cCas9 was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results.

Atg16L1, ROS and ERK regulate Cx43 in the presence of VacA. (a) Control (NC) or Atg16L1 siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43, LC3-II generation and Atg16L1 in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (b) The indicated siRNA-transfected cells were treated with toxin as shown above. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP, Atg16L1 and GAPDH in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from two experiments. Experiments were repeated two times with similar results. (c and d) AZ-521 cells were pretreated with or without 10 mM NAC (left panel) or 10 mM GSH (right panel) for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation in AZ-521 cells was performed by densitometry. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05 and **P<0.02. Experiments were repeated three times with similar results. (e) AZ-521 cells were pretreated with control (DMSO) or 10 mM NAC and then 120 nM heat-inactivated (iV) or wild-type VacA (V) for the indicated times. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. (f) AZ-521 cells were pretreated with 10 μM U0126 for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results. (g) Control (NC) or ERK siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation and ERK levels in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (h) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 4 h and total RNA was extracted as described in Materials and Methods. Cx43 mRNA was measured by real-time qPCR. Data are shown as mean±S.D. of values from two experiments. Results are shown as fold increase of GAPDH as an internal control. Experiments were repeated two times with similar results. (i) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and then reacted with anti-Cx43 antibodies (red) and incubated with DAPI (cyan). Bars represent 20 μm. Experiments were repeated three times with similar results. (j) The indicated siRNA-transfected AZ-521 cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP and cCas9 was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results.