1. Paxillin Regulates Pulmonary Arterial Smooth Muscle Cell Function in Pulmonary Hypertension 
Christine Veith, Leigh M. Marsh, Małgorzata Wygrecka, Katrin Rutschmann, Werner Seeger, Norbert Weissmann, Grażyna Kwapiszewska 
The American Journal of Pathology 
Volume 181, Issue 5, Pages (November 2012)
DOI: /j.ajpath
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
Paxillin, a novel interaction partner of Fhl-1 in murine and human lungs. A: Identification of paxillin by yeast two-hybrid analysis using N-terminal and C-terminal Fhl-1 clones (first panel, left and right, respectively). Both p53 or lamin C served as a specificity control. Schematic representation of paxillin (Prey) and Fhl-1 (Bait) proteins (second panel). Ade = adenine; aa =amino acids; His = histidine; Leu =leucine; SD = selective medium; Trp = tryptophan. B and C: Immunoprecipitation (IP) of endogenous paxillin (anti-Pxn) from (B) murine microvascular PASMC and (C) human PASMC. Western blots (WB) were probed with an anti–Fhl-1 or anti-paxillin (Pxn) antibody. IgG was used to exclude nonspecific interaction. Arrow indicates Fhl-1 (30 kDa) and paxillin (68 kDa); The asterisk indicates a heavy chain. D: Co-localization of paxillin and Fhl-1 in murine microvascular (left column) and human (right column) PASMC. E: Co-localization of paxillin and Fhl-1 in murine (left column) and human (right column) lungs. NC = isotype control staining.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
Paxillin expression is increased in lungs from IPAH patients and influenced by fibronectin. A: Real-time PCR analysis of paxillin expression in donor (control) and IPAH lung homogenates (n = 5 each). Data were analyzed by Student's t-test and are presented as mean ± SEM. B: Western blot analysis of paxillin expression in donor and IPAH lung homogenates. C: Densitometric analysis of the Western blot (n = 4 each). Data were analyzed by Student's t-test and are presented as mean ± SEM. D: Immunohistochemical staining of paxillin (in red) and α–smooth muscle actin (α-SMA; in dark red) in donor and IPAH lungs. NC = isotype control staining. E: Real-time PCR analysis of fibronectin expression in donor (control) and IPAH lung homogenates (n = 9 each). Data were analyzed by Student's t-test and are presented as mean ± SEM. F: Co-localization of fibronectin and α–smooth muscle actin (α-SMA) in donor and IPAH lungs. Phase-contrast (PC) micrographs are shown in addition. NC = isotype control staining. G: Analysis of paxillin tyrosine (Y) 118 phosphorylation and total paxillin levels in human PASMC after seeding on fibronectin-coated or uncoated plates (with or without FN) for 40 minutes (min; n = 4). H: Quantification of Western blot analysis by densitometry (n = 4 each); protein levels were normalized to β-actin. Control was set to 100%. Data were analyzed by Student's t-test and are presented as mean ± SEM. I: Co-immunoprecipitation of paxillin by Fhl-1 (anti–Fhl-1) from cell lysates of human PASMC seeded on fibronectin-coated or uncoated (with or without FN) plates. Immunoprecipitates were probed with an anti-paxillin (Pxn) or anti–Fhl-1 antibody. IgG was used to exclude nonspecific interaction. Arrow indicates paxillin (68 kDa) and Fhl-1 (30 kDa). IP = immunoprecipitation; WB = Western blot.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
PASMC adhesion is critically dependent on fibronectin, paxillin, and Fhl-1. A and B: Adhesion of human PASMC on fibronectin-coated or uncoated plates (with or without FN) assessed by immunofluorescence staining against F-actin (phalloidin) and (A) paxillin or (B) Fhl-1. C: siRNA transfection against paxillin (siPxn) or a random sequence (siR) assessed by Western blotting after 48 or 72 hours (h). D: Densitometric analysis of the Western blot analysis (n = 5). Control (siR) was set to 100%. Data were analyzed by analysis of variance followed by Dunnett's multiple-to-one comparison post hoc tests and are presented as mean ± SEM. Significant differences between control (siR) and siPxn are noted with an asterisk. E and F: Effects of paxillin (siPxn) or Fhl-1 knockdown (siFhl-1) on human PASMC adhesion determined by (E) light microscopy and (F) Crystal Violet staining (n = 6 performed in quatriplicate). Control (siR) was set to 1. Data were normalized to control and analyzed by analysis of variance followed by Dunnett's multiple-to-one comparison post hoc tests. Data are presented as mean ± SEM. Significant differences between control (siR) and siPxn or siFhl-1, respectively, are noted with an asterisk. G: Immunofluorescence staining of paxillin and phalloidin after paxillin (siPxn) or random sequence (siR) knockdown. NC = isotype control staining. H: Example of changes in paxillin distribution in human PASMC after siRNA transfection against paxillin (siPxn) or a random sequence (siR) and seeding on fibronectin-coated or uncoated plates (with or without FN) assessed by immunofluorescence staining. NC = isotype control staining.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
Paxillin and focal adhesion kinase (FAK) phosphorylation is dependent on their reciprocal expression. A: Co-localization of paxillin and FAK in focal adhesion complexes of human PASMC assessed by immunofluorescence staining. B: Representative Western blot analysis for FAK, paxillin tyrosine (Y) 118 phosphorylation and paxillin after silencing of FAK (siFAK) or a control sequence (siR) for the indicated time points. C: Densitometric quantification of the Western blot analysis at the 72-hour (h) timepoint (n = 3–7). Control (siR) was set to 100%. Data were analyzed by Student's t-test and are presented as mean ± SEM. D: Western blot for FAK tyrosine (Y) 397 or tyrosine (Y) 576, FAK and paxillin 72 hours after paxillin (siPxn) or a random sequence (siR) knockdown by siRNA transfection. E: Densitometric analysis of the Western blot analysis (n = 6–10). Control (siR) was set to 100%. Data were analyzed by Student's t-test and are presented as mean ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
Paxillin knockdown hampers proliferation and enhances apoptosis. A: Real-time PCR analysis of paxillin expression after siRNA transfection against paxillin (siPxn) in comparison to control (siR) in human PASMC (n = 5 each). Control (siR) was set to 100%. Data were analyzed by Student's t-test and are presented as mean ± SEM. B: Real-time PCR analysis of Ki-67 expression after siRNA transfection against paxillin (siPxn) in comparison to control (siR) in human PASMC (n = 5 each). Control (siR) was set to 100%. Data were analyzed by Student's t-test and are presented as mean ± SEM. C: Immunofluorescence staining of Ki-67 after paxillin silencing (siPxn) in human PASMC in comparison to control (siR). As an example, arrows indicate Ki-67–positive cells. NC = isotype control staining. D: Proliferation of human PASMC assessed by cell counting after knockdown of paxillin expression (siPxn) in comparison to control (siR, n = 8 each). Control (siR) was set to 1. Data were analyzed by Student's t-test and are presented as mean ± SEM. E: Assessment of apoptosis by caspase-3 activation after paxillin knockdown (siPxn) in comparison to control (siR, n = 10 each). Control (siR) was set to 1. Data analyzed by Student′s t-test and are presented as mean ± SEM. F: Western blot for paxillin tyrosine (Y) 118 phosphorylation, Akt serine (S) 473 phosphorylation, and Erk1/2 threonine/tyrosine (T/Y) 202/204 phosphorylation after paxillin (siPxn) or random sequence (siR) silencing. G: Phosphorylation state of Akt (left panel; n = 6) and Erk 1/2 (right panel; n = 5) quantified by densitometry; protein levels were normalized to β-actin. Control (siR) was set to 100%. Data were analyzed by Student's t-test and are presented as mean ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

7. Figure 6
Elevated paxillin expression in laser-microdissected intrapulmonary arteries from chronically hypoxic mice with PH. A: Localization of paxillin (in red) and α–smooth muscle actin (α-SMA; in dark red) in normoxic (N) and hypoxic (H) murine lung tissue. NC = isotype control staining. B: Paxillin mRNA expression in laser-microdissected normoxic (N) and hypoxic (H) murine lung vessels (outer diameter <100 μm, n = 5 each, 50 vessel profiles per animal) assessed by real-time PCR. Data were analyzed by Student's t-test and are presented as mean ± SEM. C and D: Paxillin mRNA expression assessed by real-time PCR in isolated (C) aortas or (D) Arteriae carotis from mice exposed to 1 day of normoxia (1dN) or to 1, 7, and 21 days of chronic hypoxia (1dH, 7dH, and 21dH; n = 6 each). Data were analyzed by analysis of variance followed by Dunnett's multiple-to-one comparison post hoc tests and are presented as mean ± SEM; ns = not significant.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

8. Figure 7
Hypoxia-enhanced paxillin expression in PASMC is HIF-1α dependent. A and B: Paxillin expression in human PASMC exposed to normoxic (N; 21% O2) or hypoxic (H; 1% O2) conditions for 24 hours. A: Real-time PCR (n = 6 each). Data were analyzed by Student′s t-test and are presented as mean ± SEM. B: Western blot analysis. C: Densitometric analysis of the Western blot (n = 4 each). Data were analyzed by Student's t-test and are presented as mean ± SEM. D: Paxillin expression in human PASMC after normoxic (N; 21% O2) or hypoxic (H; 1% O2) exposure assessed by immunofluorescence staining. NC = isotype control staining. E: Paxillin promoter analysis. Potential hypoxia response elements (HRE) in promoter (1500-bp) region are shown. The coding sequence of the paxillin gene is marked with + 1. F: Paxillin mRNA expression in human PASMC after siRNA transfection against HIF-1α (siHIF-1α) on hypoxic exposure (24 hours 1% O2, 24 hours H) in comparison to control (siR, n = 5 each). Data were analyzed by Student's t-test and are presented as mean ± SEM. G: Representative electrophoretic mobility shift assays (EMSA) for each hypoxia response element (HRE) using normoxic (2 hours, 21% O2) and hypoxic (H; 2 hours, 1% O2) nuclear extracts (NE) from human PASMC and biotin-labeled or unlabeled (Cc = cold competitor) probes; n = 3 for each HRE. Arrow indicates shift complex. H: Chromatin immunoprecipitation was performed from hypoxic (2 hours, 1% O2) human PASMC using an HIF-1α (anti–HIF-1α) or an IgG antibody for precipitation. Representative agarose gel electrophoresis of PCR products amplified using specific primers for hypoxia response elements (HREs) in the paxillin promoter region are shown (n = 2–3 for each HRE). bp = Base pairs; input = input control. Arrow indicates size of product.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions