Journal: Molecular Psychiatry

Article Title: PAI-1 protein is a key molecular effector in the transition from normal to PTSD-like fear memory

doi: 10.1038/s41380-021-01024-1

Figure Lengend Snippet: PAI-1 mRNA, measured by qPCR ( a ), and protein expressions, measured by western blot ( b , c ), in response to 100 and 1000 nM of Cort for 3 h (180 min) in PC12 cells. PAI-1, tPA, P-TrkB, and P-Erk1/2 MAPK proteins measured by western blot ( d , e ) in dorsal hippocampal slices of Sprague-Dawley rats incubated with 10 and 1000 nM of Cort for 1 h (60 min) and 3 h (180 min). α-tubulin and βIII-tubulin were used as a loading control. X-ray films were quantified by densitometry (OD). Dunnett’s multiple comparisons post hoc test after ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.005 compared to control conditions. Plotted values are means ± sem.

Article Snippet: Rabbit polyclonal anti-PAI-1 antibodies were from Lifespan Biosciences (LSBio#C81062, 1/1000, WA, USA) and Epitomics (#3917-1, 1/3000, CA, USA), anti-tPA (#T5600-05G; 1/5000) was from US Biological (MA, USA), anti-Erk1/2 MAPK (#06-182; 1/50000) was from Millipore (MA, USA), and anti-Phospho-Erk1/2 MAPK (#9101S; 1/1000) was from CST (MA, USA).

Techniques: Western Blot, Incubation

Journal: Molecular Psychiatry

Article Title: PAI-1 protein is a key molecular effector in the transition from normal to PTSD-like fear memory

doi: 10.1038/s41380-021-01024-1

Figure Lengend Snippet: Plasma corticosterone levels ( a ), PAI-1, tPA, and P-Erk1/2 MAPK proteins measured by western blot ( b , c ) in the dorsal hippocampus of C57BL/6J mice in response to 30 min, 1 h (60 min), and 3 h (180 min) restraint stress. βIII-tubulin was used as a loading control. X-ray films were quantified by densitometry (OD). Dunnett’s multiple comparisons post hoc test after ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.005 compared to control conditions. Plotted values are means ± sem.

Article Snippet: Rabbit polyclonal anti-PAI-1 antibodies were from Lifespan Biosciences (LSBio#C81062, 1/1000, WA, USA) and Epitomics (#3917-1, 1/3000, CA, USA), anti-tPA (#T5600-05G; 1/5000) was from US Biological (MA, USA), anti-Erk1/2 MAPK (#06-182; 1/50000) was from Millipore (MA, USA), and anti-Phospho-Erk1/2 MAPK (#9101S; 1/1000) was from CST (MA, USA).

Techniques: Western Blot

Journal: Molecular Psychiatry

Article Title: PAI-1 protein is a key molecular effector in the transition from normal to PTSD-like fear memory

doi: 10.1038/s41380-021-01024-1

Figure Lengend Snippet: Fear responses, expressed as percent of time spent freezing, 24 h after conditioning in C57BL/6J mice exposed in a safe environment to the tone not predicting the threat (nonpredicting cue, a and b ) or to the environment in which the conditioning was performed (predicting context, c and d ). Expression of P-Erk1/2 MAPK ( e , f ) and PAI-1 ( g , h ) proteins in the dorsal hippocampus at different times after the conditioning sessions ( e , g ) and expressed as area under the curve encompassing the 24 h of analysis ( f , h ). Example of the western blot used for protein quantification by densitometry after normalization with the level of βIII-tubulin ( i ). Immediately after the conditioning session animals received an injection of either vehicle (Veh; NaCl 0.9% i.p., white symbol) or Cort (2 mg/kg i.p., black symbol). Gray symbol: control animals that were manipulated but not exposed to conditioning. Magnitude of tone conditioning represented by a normalized ratio: (tone − ((pre + post)/2))/(tone + ((pre + post)/2)) ( b ). Student’s t test and Sidak’s and Bonferroni/Dunn’s multiple comparisons post hoc test after ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.005 compared to Veh group. Plotted values are means ± sem.

Article Snippet: Rabbit polyclonal anti-PAI-1 antibodies were from Lifespan Biosciences (LSBio#C81062, 1/1000, WA, USA) and Epitomics (#3917-1, 1/3000, CA, USA), anti-tPA (#T5600-05G; 1/5000) was from US Biological (MA, USA), anti-Erk1/2 MAPK (#06-182; 1/50000) was from Millipore (MA, USA), and anti-Phospho-Erk1/2 MAPK (#9101S; 1/1000) was from CST (MA, USA).

Techniques: Expressing, Western Blot, Injection

Journal: Molecular Psychiatry

Article Title: PAI-1 protein is a key molecular effector in the transition from normal to PTSD-like fear memory

doi: 10.1038/s41380-021-01024-1

Figure Lengend Snippet: Fear responses, expressed as percent of time spent freezing, 24 h after conditioning in C57BL/6J mice exposed in a safe environment to the tone not predicting the threat (nonpredicting cue, a and c ) or to the environment in which the conditioning was performed (predicting context, b and d ). Immediately after the conditioning session animals received one of the following treatments: an injection of vehicle (Veh; NaCl 0.9% i.p., white symbol); an injection of Cort (2 mg/kg i.p., black symbol); an intrahippocampal infusion of PAI-1 (30, 90, or 240 ng/side, symbols with different shadows of blue). Magnitude of tone conditioning represented by a normalized ratio: (tone − ((pre + post)/2))/(tone + ((pre + post)/2)) ( c ). Bonferroni/Dunn’s multiple comparisons post hoc test after ANOVA: * p < 0.05, *** p < 0.005 compared to Veh/Veh group. Plotted values are means ± sem.

Article Snippet: Rabbit polyclonal anti-PAI-1 antibodies were from Lifespan Biosciences (LSBio#C81062, 1/1000, WA, USA) and Epitomics (#3917-1, 1/3000, CA, USA), anti-tPA (#T5600-05G; 1/5000) was from US Biological (MA, USA), anti-Erk1/2 MAPK (#06-182; 1/50000) was from Millipore (MA, USA), and anti-Phospho-Erk1/2 MAPK (#9101S; 1/1000) was from CST (MA, USA).

Techniques: Injection

Journal: Molecular Psychiatry

Article Title: PAI-1 protein is a key molecular effector in the transition from normal to PTSD-like fear memory

doi: 10.1038/s41380-021-01024-1

Figure Lengend Snippet: Fear responses, expressed as percent of time spent freezing, 24 h after conditioning in C57BL/6J mice exposed in a safe environment to the tone not predicting the threat (nonpredicting cue, a , b , e , and f ) or to the environment in which the conditioning was performed (predicting context, c , d , g , and h ). Immediately after the conditioning session animals received one of the following treatments: an injection of (white symbol) vehicle (Veh; NaCl 0.9% i.p.) alone or in combination with the intrahippocampal infusion of either (blue symbol) PAI-1 (240 ng/side) or (orange symbol) PAI-1 (240 ng/side) + mature BDNF (100 ng/side); an injection of (black symbol) Cort alone (2 mg/kg i.p.) or in combination with the intrahippocampal infusion of either the PAI-1 antagonist tiplaxtinin (5 ng/side, green symbol) or the vehicle of tiplaxtinin (gray symbol). Magnitude of tone conditioning represented by a normalized ratio: (tone − ((pre + post)/2))/(tone + ((pre + post)/2)) ( b and f ). Bonferroni/Dunn’s multiple comparisons post hoc test after ANOVA: ** p < 0.001 and *** p < 0.005 vs Veh/Veh group, # p < 0.05 and ### p < 0.005 compared to Veh/PAI-1 and Cort groups. Plotted values are means ± sem.

Article Snippet: Rabbit polyclonal anti-PAI-1 antibodies were from Lifespan Biosciences (LSBio#C81062, 1/1000, WA, USA) and Epitomics (#3917-1, 1/3000, CA, USA), anti-tPA (#T5600-05G; 1/5000) was from US Biological (MA, USA), anti-Erk1/2 MAPK (#06-182; 1/50000) was from Millipore (MA, USA), and anti-Phospho-Erk1/2 MAPK (#9101S; 1/1000) was from CST (MA, USA).

Techniques: Injection

Journal: Scientific Reports

Article Title: Requirement of HDAC6 for activation of Notch1 by TGF-β1

doi: 10.1038/srep31086

Figure Lengend Snippet: ( A ) Western analysis of cytoplasmic and nuclear fractions demonstrating nuclear translocation Notch1 ICN after treatment with TGFβ-1. ( B,C ) qPCR analysis of Notch signaling genes HEY-1 ( B ) and HES-1 ( C ) in A549 cells treated with 2.5 ng/mL TGFβ-1 for 24 hours. The –fold change of each transcript was obtained by setting the value of the unexposed cells to 1. ( D , E ) Western analysis of Notch signaling genes HES-1 ( D ) and TGF-β1 responsive gene PAI-1, and HEY-1 ( E ) in A549 cells two separate blots of the same experiment treated with 2.5 ng/mL TGFβ-1 for a time course over 48 hours. ( F , G ) Densitometry analysis of three independent experiments and probed for HES-1 ( F ) and HEY-1 ( G ). Data for qPCR presented as mean +/− SEM of triplicate wells and are representative of three independent experiments statistically analyzed using one-way ANOVA. Data for densitometry analysis presented as +/− STD of three independent experiments statistically analyzed using unpaired T-tests. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the relative control.

Article Snippet: Anti-PAI1 rabbit polyclonal antibody (500-P260) was purchased from PeproTech (Rocky Hill, NJ, USA) and was used for western analysis at 1:2,000.

Techniques: Western Blot, Translocation Assay, Control

Journal: Scientific Reports

Article Title: Requirement of HDAC6 for activation of Notch1 by TGF-β1

doi: 10.1038/srep31086

Figure Lengend Snippet: ( A ) Serum-starved A549 variants were exposed to TGF-β1 (2.5 ng/ml) for the indicated duration, cell lysates were fractionated to cytoplasmic and nuclear extracts and protein levels of nuclear ICN and HDAC6 were examined by Western analysis. ( B ) RNA was isolated from A549 variants exposed to TGF-β1 (2.5 ng/ml) for 24 hours. Quantitative RT-PCR was carried out for HEY-1. ( C ) Same experimental conditions as in B, except the transcript of interest examined was HES-1. The –fold change of each transcript was obtained by setting the value of the unexposed pS cells to 1 ( D ) Experimental conditions and fractionation of serum starved A549 variant cell lysates were the same as in A, except the duration of TGF-β1 treatment was for 24 and 48 hours. Protein levels of HEY-1 and HES-1 in the nuclear extract were examined by western analysis. ( E ) Cytoplasmic fraction of the same experiment as D, except TGF-β1 responsive genes PAI-1 and Vimentin protein levels were examined by western analysis. ( F ) Densitometry analysis of western blots from three independent experiments represented in panel ( D ); levels of HEY-1 and HES-1 were relativized to lamin A/C. For qPCR analysis data presented as mean +/− SEM of triplicate wells and are representative of three independent experiments statistically analyzed using one-way ANOVA. For densitometry analysis data presented as mean +/− STD of three independent experiments statistically analyzed using one-way ANOVA. *P <0.05, **P < 0.01, and ***P < 0.001 compared with the relative control.

Article Snippet: Anti-PAI1 rabbit polyclonal antibody (500-P260) was purchased from PeproTech (Rocky Hill, NJ, USA) and was used for western analysis at 1:2,000.

Techniques: Western Blot, Isolation, Quantitative RT-PCR, Fractionation, Variant Assay, Control

Journal: Scientific Reports

Article Title: Requirement of HDAC6 for activation of Notch1 by TGF-β1

doi: 10.1038/srep31086

Figure Lengend Snippet: ( A – C ) Serum-starved A549 cells were pre-treated for six hours with either 1 μM or 10 μM 17-AAG or an equivalent volume of DMSO for six hours before being exposed to TGF-β1 (2.5 ng/ml) for 24 hours. RNA was isolated and quantitative RT-PCR was carried out for HEY-1, HES-1, and PAI1 transcripts. ( D ) A549 cells transiently transfected with siRNA targeting HDAC6, Notch1, or non-specific control were serum starved and treated with TGF-β1 for 24 hours. RNA was isolated and Quantitative RT-PCR was carried out for HMOX1 transcripts. The –fold change of each transcript was obtained by setting the value of the untreated cells to 1. Data presented as mean +/− SEM of triplicate wells and are representative of three independent experiments ( A – C ) and D as mean +/− STD of triplicate wells from a single independent experiment statistically analyzed using one-way ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the relative control.

Article Snippet: Anti-PAI1 rabbit polyclonal antibody (500-P260) was purchased from PeproTech (Rocky Hill, NJ, USA) and was used for western analysis at 1:2,000.

Techniques: Isolation, Quantitative RT-PCR, Transfection, Control