pegfp-c2 senp2 Search Results


plasmids encoding pegfp c2 senp2  (Addgene inc)
91
Addgene inc plasmids encoding pegfp c2 senp2
A. Immunoblot analysis of Ubc9 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Ubc9 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. B. Immunoblot analysis of Sae1, Sae2 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Sae1 and Sae2 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. C. Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS), SENP1 or <t>SENP2</t> siRNA-transfected A549 cells infected with Kp52145 for 5 h. AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. D.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localised in either nucleus or cytoplasm is represented on the graph and is the result of independent counting of 100 cells from each of 3 independent experiments. E.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). F.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were treated with the proteasomal inhibitor MG262 (5 μM, 2 h before infection), the nuclear export inhibitor Leptomycin B (LMB, 10 nM, 2 h before infection) or DMSO (vehicle solution) and infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localized in either nucleus or cytoplasm is represented on the graphs and is the result of independent counting of 100 cells from each of 3 independent experiments. G.Immunoblot analysis of SUMO1 and tubulin levels in lysates of the nuclear export inhibitor Leptomycin B (LMB, 5 μM, 2 h before infection) or DMSO (vehicle solution)-treated A549 infected with Kp52145 for 5 h or left uninfected (n.i.). SUMO1 smears were quantified using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to DMSO non-infected control cells. **P ≤ 0.01 versus DMSO n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. H.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of A549 infected with Kp52145 for 5 h. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. I.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of control (AS), Rbx1, Skp1, Cul-1, βTrCP or Skp2 siRNA-transfected A549 cells left uninfected (n.i.). J.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). Lysates of A549 treated with 5 mM of H 2 O 2 for 10 min were used as a positive control for Cullin-1 deNEDDylation. K.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. L.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of control (AS) or CSN5 siRNA-transfected A549. Cells were transfected with a SENP2-FLAG plasmid 24 h after the siRNA transfection and infected the following day with Kp52145 for 5 h or left uninfected. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. M.Immunoblot analysis of Senp2 and tubulin levels in cytosolic extracts of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. N.Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. **P ≤ 0.01 versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. In all panels data are representative of at least three independent experiments.
Plasmids Encoding Pegfp C2 Senp2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmids encoding pegfp c2 senp2/product/Addgene inc
Average 91 stars, based on 1 article reviews
plasmids encoding pegfp c2 senp2 - by Bioz Stars, 2026-03
91/100 stars
  Buy from Supplier

Image Search Results


A. Immunoblot analysis of Ubc9 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Ubc9 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. B. Immunoblot analysis of Sae1, Sae2 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Sae1 and Sae2 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. C. Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS), SENP1 or SENP2 siRNA-transfected A549 cells infected with Kp52145 for 5 h. AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. D.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localised in either nucleus or cytoplasm is represented on the graph and is the result of independent counting of 100 cells from each of 3 independent experiments. E.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). F.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were treated with the proteasomal inhibitor MG262 (5 μM, 2 h before infection), the nuclear export inhibitor Leptomycin B (LMB, 10 nM, 2 h before infection) or DMSO (vehicle solution) and infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localized in either nucleus or cytoplasm is represented on the graphs and is the result of independent counting of 100 cells from each of 3 independent experiments. G.Immunoblot analysis of SUMO1 and tubulin levels in lysates of the nuclear export inhibitor Leptomycin B (LMB, 5 μM, 2 h before infection) or DMSO (vehicle solution)-treated A549 infected with Kp52145 for 5 h or left uninfected (n.i.). SUMO1 smears were quantified using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to DMSO non-infected control cells. **P ≤ 0.01 versus DMSO n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. H.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of A549 infected with Kp52145 for 5 h. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. I.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of control (AS), Rbx1, Skp1, Cul-1, βTrCP or Skp2 siRNA-transfected A549 cells left uninfected (n.i.). J.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). Lysates of A549 treated with 5 mM of H 2 O 2 for 10 min were used as a positive control for Cullin-1 deNEDDylation. K.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. L.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of control (AS) or CSN5 siRNA-transfected A549. Cells were transfected with a SENP2-FLAG plasmid 24 h after the siRNA transfection and infected the following day with Kp52145 for 5 h or left uninfected. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. M.Immunoblot analysis of Senp2 and tubulin levels in cytosolic extracts of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. N.Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. **P ≤ 0.01 versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. In all panels data are representative of at least three independent experiments.

Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: A. Immunoblot analysis of Ubc9 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Ubc9 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. B. Immunoblot analysis of Sae1, Sae2 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Sae1 and Sae2 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. C. Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS), SENP1 or SENP2 siRNA-transfected A549 cells infected with Kp52145 for 5 h. AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. D.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localised in either nucleus or cytoplasm is represented on the graph and is the result of independent counting of 100 cells from each of 3 independent experiments. E.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). F.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were treated with the proteasomal inhibitor MG262 (5 μM, 2 h before infection), the nuclear export inhibitor Leptomycin B (LMB, 10 nM, 2 h before infection) or DMSO (vehicle solution) and infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localized in either nucleus or cytoplasm is represented on the graphs and is the result of independent counting of 100 cells from each of 3 independent experiments. G.Immunoblot analysis of SUMO1 and tubulin levels in lysates of the nuclear export inhibitor Leptomycin B (LMB, 5 μM, 2 h before infection) or DMSO (vehicle solution)-treated A549 infected with Kp52145 for 5 h or left uninfected (n.i.). SUMO1 smears were quantified using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to DMSO non-infected control cells. **P ≤ 0.01 versus DMSO n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. H.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of A549 infected with Kp52145 for 5 h. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. I.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of control (AS), Rbx1, Skp1, Cul-1, βTrCP or Skp2 siRNA-transfected A549 cells left uninfected (n.i.). J.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). Lysates of A549 treated with 5 mM of H 2 O 2 for 10 min were used as a positive control for Cullin-1 deNEDDylation. K.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. L.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of control (AS) or CSN5 siRNA-transfected A549. Cells were transfected with a SENP2-FLAG plasmid 24 h after the siRNA transfection and infected the following day with Kp52145 for 5 h or left uninfected. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. M.Immunoblot analysis of Senp2 and tubulin levels in cytosolic extracts of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. N.Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. **P ≤ 0.01 versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. In all panels data are representative of at least three independent experiments.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid # 13382) and infected 24 h later.

Techniques: Western Blot, Infection, Modification, Control, Transfection, Fluorescence, Microscopy, Staining, Immunoprecipitation, Negative Control, Positive Control, Plasmid Preparation

A. ELISA of IL-8 secreted by A549 cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1), which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. B. ELISA of IL-8 secreted by control (AS) or Senp2 siRNA-transfected A549, which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus AS determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. C.ELISA of TNFα secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. D.ELISA of TNFα secreted by antagomir-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus Negative Kp52145 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. E.Immunoblot analysis of IκBα and tubulin levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. F.Immunoblot analysis of JNK (P-JNK), ERK (P-ERK) and p38 (P-p38) phosphorylation levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. Tubulin immunoblotting was used as the loading control. n.i. – non-infected control G.ELISA of IL-8 or TNFα secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected A549 or MH-S respectively, which were treated with the NF-κB inhibitor BAY 11-7082 (5 μM, 2 h before infection) or DMSO (vehicle solution). Cells were left untreated (n.i.) or infected with Kp52145 for 1 h (MH-S) or 3 h (A549) of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. H.Percentage of intracellular survival in MH-S cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1). Cells were infected with Kp52145 for 30 min (MOI 100:1), wells were washed and incubated with medium containing gentamicin (100 μg mL -1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution and viable counting on LB agar plates. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering pcDNA3 as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using unpaired t-test with correction for Holm-Sidak’s multiple comparisons test. I.Percentage of intracellular survival in antagomir transfected MH-S cells. Cells were infected with Kp52145 for 30 min (MOI 100:1), wells were washed and incubated with medium containing gentamicin (100 μg mL -1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution and viable counting on LB agar plates. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering the negative control as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. **P ≤ 0.01; *P ≤ 0.05; versus negative control determined using one way-ANOVA with Holm-Sidak’s multiple comparisons test. In panels E and F, data are representative of at least three independent experiments.

Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: A. ELISA of IL-8 secreted by A549 cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1), which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. B. ELISA of IL-8 secreted by control (AS) or Senp2 siRNA-transfected A549, which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus AS determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. C.ELISA of TNFα secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. D.ELISA of TNFα secreted by antagomir-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus Negative Kp52145 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. E.Immunoblot analysis of IκBα and tubulin levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. F.Immunoblot analysis of JNK (P-JNK), ERK (P-ERK) and p38 (P-p38) phosphorylation levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. Tubulin immunoblotting was used as the loading control. n.i. – non-infected control G.ELISA of IL-8 or TNFα secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected A549 or MH-S respectively, which were treated with the NF-κB inhibitor BAY 11-7082 (5 μM, 2 h before infection) or DMSO (vehicle solution). Cells were left untreated (n.i.) or infected with Kp52145 for 1 h (MH-S) or 3 h (A549) of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. H.Percentage of intracellular survival in MH-S cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1). Cells were infected with Kp52145 for 30 min (MOI 100:1), wells were washed and incubated with medium containing gentamicin (100 μg mL -1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution and viable counting on LB agar plates. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering pcDNA3 as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using unpaired t-test with correction for Holm-Sidak’s multiple comparisons test. I.Percentage of intracellular survival in antagomir transfected MH-S cells. Cells were infected with Kp52145 for 30 min (MOI 100:1), wells were washed and incubated with medium containing gentamicin (100 μg mL -1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution and viable counting on LB agar plates. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering the negative control as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. **P ≤ 0.01; *P ≤ 0.05; versus negative control determined using one way-ANOVA with Holm-Sidak’s multiple comparisons test. In panels E and F, data are representative of at least three independent experiments.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid # 13382) and infected 24 h later.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Control, Plasmid Preparation, Infection, Bacteria, Incubation, Western Blot, Phospho-proteomics, Lysis, Serial Dilution, Negative Control

Working model of K. pneumoniae strategies to decrease SUMOylation in epithelial cells and macrophages. In epithelial cells, Kp52145 activates the signalling pathway EGFR-PI3K-AKT-ERK-GSK3β to increase the COP9 signalosome component CSN5 which inhibits NEDDylation of Cullin-1 and prevents proteasomal degradation of SENP2. SENP2 then accumulates in the cytosol preventing SUMOylation. In macrophages, Kp52145 via TLR4-TRAM-TRIF induces the production of type-I interferon, which signals through the IFNAR1 receptor. Type-I interferon stimulates transcription of the miRNA let-7 which prevents SUMOylation. Both strategies lead to increased intracellular survival and subversion of host responses.

Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: Working model of K. pneumoniae strategies to decrease SUMOylation in epithelial cells and macrophages. In epithelial cells, Kp52145 activates the signalling pathway EGFR-PI3K-AKT-ERK-GSK3β to increase the COP9 signalosome component CSN5 which inhibits NEDDylation of Cullin-1 and prevents proteasomal degradation of SENP2. SENP2 then accumulates in the cytosol preventing SUMOylation. In macrophages, Kp52145 via TLR4-TRAM-TRIF induces the production of type-I interferon, which signals through the IFNAR1 receptor. Type-I interferon stimulates transcription of the miRNA let-7 which prevents SUMOylation. Both strategies lead to increased intracellular survival and subversion of host responses.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid # 13382) and infected 24 h later.

Techniques: