Supplementary Materials

Supplementary Material for:

NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension

Andriy O. Samokhin, Thomas Stephens, Bradley M. Wertheim, Rui-Sheng Wang, Sara O. Vargas, Lai-Ming Yung, Minwei Cao, Marcel Brown, Elena Arons, Paul B. Dieffenbach, Jason G. Fewell, Majed Matar, Frederick P. Bowman, Kathleen J. Haley, George A. Alba, Stefano M. Marino, Rahul Kumar, Ivan O. Rosas, Aaron B. Waxman, William M. Oldham, Dinesh Khanna, Brian B. Graham, Sachiko Seo, Vadim N. Gladyshev, Paul B. Yu, Laura E. Fredenburgh, Joseph Loscalzo, Jane A. Leopold, Bradley A. Maron*

*Corresponding author. Email: bmaron{at}bwh.harvard.edu

Published 13 June 2018, Sci. Transl. Med. 10, eaap7294 (2018)
DOI: 10.1126/scitranslmed.aap7294

This PDF file includes:

  • Materials and Methods
  • Fig. S1. Fibrosome and ALDO-fibrosome networks.
  • Fig. S2. Computational modeling predicts that NEDD9-Cys18 is highly prone to oxidant stress.
  • Fig. S3. Proteasomal inhibition or oxidant stress increases NEDD9 accumulation in HPAECs.
  • Fig. S4. ALDO does not affect NEDD9 expression in cell types associated with adaptive fibrosis.
  • Fig. S5. NEDD9 is expressed in the nucleus of HPAECs and PAH-HPAECs.
  • Fig. S6. NKX2-5 and NEDD9 colocalize to the HPAEC nucleus, and inhibition of NKX2-5 prevents COL3A1 mRNA transcription mediated by ALDO.
  • Fig. S7. ALDO increases NEDD9-dependent fibrosis in HPAECs without changes to EndMT marker expression.
  • Fig. S8. Nuclear NEDD9 expression in vascular cells from pulmonary arterioles in human PAH.
  • Fig. S9. The cysteine sulfonic acid modification at NEDD9-Cys18 is observed in PAH-HPAECs.
  • Fig. S10. NEDD9 expression is increased globally in remodeled pulmonary arterioles from patients with PAH.
  • Fig. S11. The exosome marker CD9 in cells treated with exosomes from HPAECs.
  • Fig. S12. NEDD9 was not detected by LC-MS in the culture medium of ALDO-treated cells.
  • Fig. S13. Inhibition of NEDD9 improves vascular fibrosis in PAH in vivo.
  • Table S1. The effect of NEDD9 inhibition on differential expression of genes related to fibrosis in HPAECs.
  • Table S2. Clinical and demographic information for biological samples used in this study.
  • Legends for movies S1 to S3
  • References (4165)

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Other Supplementary Material for this manuscript includes the following:

  • Movie S1 (.avi format). Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in vehicle-treated HPAECs.
  • Movie S2 (.avi format). Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in ALDO-treated HPAECs.
  • Movie S3 (.avi format). Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in PAH-HPAECs.
  • Data file S1 (Microsoft Excel format). Differentially expressed genes from RNASEQ analysis of HPAECs treated with ALDO (10−7 M) for 24 hours versus ALDO for 24 hours after transfection with si-NEDD9.
  • Data file S2 (.csv format). Differentially expressed genes from RNASEQ analysis of HPAECs treated with vehicle control or ALDO (10−7 M) for 24 hours.
  • Data file S3 (.csv format). Differentially expressed genes from RNASEQ analysis of HPAECs treated with vehicle control or ALDO (10−7 M) for 24 hours after transfection with si-NEDD9.
  • Data file S4 (Microsoft Excel format). Primary data.

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