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Page 4 of 11 Narayanan et al. J Cancer Metastasis Treat 2019;5:36 I http://dx.doi.org/10.20517/2394-4722.2018.77

confocal laser-scanning microscope (Olympus) was used for IF IH-stained slides and the images were
processed with CellSens Dimension 1.11 software using the 2D deconvolution algorithm (Olympus).

NanoString mRNA expression analysis
Six snap-frozen LMCA tissue samples from the original cohort of 16 patients included for DAB IHC staining
were used to isolate total RNA. RNA was extracted using the MagJET RNA Kit and KingFisher Duo
(ThermoFisher Scientific) protocol and quantitated by the NanoDrop 2000 Spectrophotometer (Thermo
Scientific) and Qubit (Thermo Scientific). mRNA was assayed by New Zealand Genomics Ltd (Dunedin,
NZ), using the NanoString nCounter Gene Expression Assay (NanoString Technologies, Seattle, WA, USA).
Probes for the genes were designed and synthesized by NanoString Technologies and are PRR (ATP6AP2,
NM_005765.2), ACE (CD143, NM-000789.2), ATIIR1 (AGTR1, NM_000685.3), and ATIIR2 (AGTR2,
NM_000686.3). Raw data were analyzed by nSolver software (NanoString Technologies) using standard
settings and were normalized against the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), as routinely performed in our laboratory [16,28] .

Western blotting
Total protein was extracted from three snap-frozen LMCA tissue samples from the original cohort of 16
patients used for DAB IHC staining, resolved by SDS-PAGE, and transferred to a PVDF membrane as
[31]
previously described , and probed using the following antibodies: PRR (1:500, cat# ab40790, Abcam),
ATIIR1 (1:500; cat# sc-1173, Santa Cruz, CA, USA), ATIIR2 (1:1000; cat# ab92445, Abcam), ACE (1:200; cat#
sc-12184, Santa Cruz), and β-actin (1:500; cat# ab8229, Abcam). Secondary and tertiary antibodies were goat
TM
anti-rabbit HRP (1:2000; cat# ab6721, Abcam) for PRR and ATIIR1, rabbit anti-goat Superclonal biotin
TM
conjugate (1:5000; cat# A27013, Thermo Fisher) and goat anti-rabbit Superclonal biotin conjugate (1:5000;
TM
cat# A27035, Thermo Fisher) followed by Pierce Streptavidin Poly-HRP (1:5000, cat# 21140, Thermo
Fisher) for ACE and ATIIR2, respectively, and chicken anti-goat Alexa Fluor 647 (1:2000; cat# A21244, Life
Technologies) for β-actin. Clarity Western ECL (cat# 1705061, Bio-Rad) was used as the substrate for HRP
bands and the Chemi Doc MP Imaging System (Bio-Rad) and ImageLab 5.0 software (Bio-Rad) were used
for detection and analysis, as previously used [16,28] . Positive controls were snap-frozen human placenta tissue
for PRR and ATIIR1, snap-frozen mouse lung tissue for ACE, and a recombinant ATIIR2 protein (cat#
H00000186-P01, Novus Biologicals) for ATIIR2. Matched mouse (1:500; cat# ab18443, Abcam) and rabbit
(1:500; cat# ab171870, Abcam) isotype controls were used as appropriate negative controls.

Statistical analysis
Statistical analysis of the NanoString mRNA data was performed using t test (SPSS v 24).

RESULTS
Histology and DAB IHC staining
H&E staining confirmed the presence of LMCA on the slides for each of the 16 tissue samples. DAB IHC
staining demonstrated the expression of PRR [Figure 1A, brown] which was localized to cells within the
TNs, cells within the PTS and the endothelium of the microvessels within the PTS. ACE [Figure 1B, brown]
was expressed on the luminal surface of the TNs and weakly on the endothelium of the microvessels within
the PTS. Strong cytoplasmic expression of ATIIR1 [Figure 1C, brown] was present on the cells within the
TNs, the cells within the PTS and the endothelium of the microvessels within the PTS. ATIIR2 [Figure 1D,
brown] was also expressed by the cells within the TNs, and weakly on the endothelium of the microvessels
within the PTS.

The expected expression patterns of positive controls for PRR [Supplementary Figure 1A, brown],
ACE [Supplementary Figure 1B, brown], ATIIR1 [Supplementary Figure 1C, brown], and ATIIR2
[Supplementary Figure 1D, brown] were demonstrated in human placenta, kidney, liver and kidney,