Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: Immunohistochemical staining for ( A ) Beclin1, ( B ) LC3B, and ( C ) p62 as markers of autophagy in combination with SMA, Cx40, and DAPI in lungs of human control and IPAH patients ( n = 4). Closeup of boxed region in A is shown to the right. Scale bars: 50 μm.

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Immunohistochemical staining, Staining

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: ( A ) qRT-PCR analysis of autophagy-related gene products and HIF1A , HIF2A , and PDGFB from lung lysates of patients with IPAH compared with that of control individuals ( n = 6). ( B ) Western blots of lung lysates of IPAH patients and controls probed for Beclin1, LC3B, p62, and GAPDH. ( C ) Densitometry of protein bands shown in B relative to GAPDH and normalized to control ( n = 4). ( D ) Acta2-CreER T2 ROSA26R Zs/+ mice were induced with tamoxifen, rested for 5 days, exposed to normoxia or hypoxia for 21 days or to hypoxia for 21 days, followed by re-normoxia for 10 days. Lung Zs + cells were isolated by FACS, and the expression of autophagy genes Atg5 , Atg7 , Becn1 , and Map1lc3b was analyzed by qRT-PCR and normalized to normoxia. n = 3 mice (1 male, 2 females) per experimental group. Significance assessed by 2-tailed Student’s t test ( A and C ) or multifactor ANOVA with Tukey’s multiple-comparison test ( D ).

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Expressing, Comparison

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: ( A ) Experimental strategy for B . ( B ) Human PAECs were exposed to normoxia or hypoxia (3% O 2 ) for 16 hours, and the conditioned medium was collected and pretreated with either anti–PDGF-B blocking antibody or IgG isotype control for 1 hour. Human PASMCs were incubated with the pretreated PAEC-conditioned medium in normoxic conditions for 48 hours, and then qRT-PCR was used to assess mRNA levels of ATG5 , ATG7 , BECN1 , and MAP1LC3B in the PASMCs. Transcript levels relative to 18S rRNA were normalized to normoxia PAEC medium treated with IgG (dashed line). n = 3. Significance assessed by multifactor ANOVA with Tukey’s multiple-comparison test. ( C ) Experimental strategy for D and E . ( D ) Cdh5-CreER T2 Pdgfb fl/fl mice were exposed to hypoxia for 35 days and tamoxifen (1 mg/day) was or was not administered on hypoxia days 17–21. Vibratome lung sections were stained for Beclin1, SMA, and nuclei (DAPI). Closeups of boxed region are shown below. Arrowheads indicate Beclin1 + SMA + cells. ( E ) Quantification of the percentage of distal arteriole SMCs that are Beclin1 + . n = 3 mice (2 males, 1 female) per experimental group, 3–4 arterioles analyzed per mouse. Significance assessed by 2-tailed Student’s t test. Scale bar: 20 μm.

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Blocking Assay, Incubation, Quantitative RT-PCR, Comparison, Staining

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: ( A ) Experimental strategy for B – D . ( B ) Acta2-CreER T2 ROSA26R Zs/+ mice were exposed to hypoxia or normoxia for 31 days and STI571 was administered at 0, 50, or 100 mg/kg/d by daily intraperitoneal injections on hypoxia days 21–31. Lung Zs + SMCs were isolated by FACS, and expression levels of Atg5 , Atg7 , Becn1 , and Map1lc3b with hypoxia relative to normoxia, no STI571 were analyzed by qRT-PCR. n = 3 mice (2 males, 1 female) per experimental group. ( C ) Vibratome lung sections were stained for Beclin1, SMA, and nuclei (DAPI). Closeups of boxed regions are shown below. ( D ) Quantification of the percentage of distal arteriole SMCs that are Belclin1 + . n = 3 mice (1 male, 2 female) per experimental group, 3–4 arterioles analyzed per mouse. Significance assessed by multifactor ANOVA with Tukey’s multiple-comparison test ( B and D ). Scale bar: 20 μm.

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Staining, Comparison