Page 81 - GTM-4-2

Global Translational Medicine Angiotensinogen in liver steatosis

and counterstained in Mayer’s hematoxylin (#26043-06, tests for normality or homogeneity of variance were
Electron Microscopy Sciences, USA) for 30 s. Coverslips analyzed by Kruskal–Wallis one-way ANOVA on Ranks
were applied with warmed glycerol gelatin (#GG1, with Dunn’s method. p<0.05 was considered statistically
Millipore Sigma). Representative microscopic images were significant.
captured using an Eclipse NI microscope with a DS-Ri2 Bulk RNA sequencing data were analyzed on R
camera (Nikon, Japan). For quantification of Oil Red O, (v4.1.0). 21,22 Ensembl gene identifiers with no detected read
whole liver sections were imaged using a Z7 slide scanner counts in any sample were excluded. Read count data were
(Zeiss, Germany). Oil Red O staining was quantified using normalized using the TMM method in edgeR package
an RGB color threshold (red: 0 – 255, green: 0 – 255, blue: (v3.36.0) to adjust for biases in library size and composition.
0 – 185) and normalized against total tissue area, defined P-values were calculated using either “glmQLFTest” for
by total pixels – white background pixels (red: 250 – 255, multi-group or “exactTest” for two-group comparisons in
green: 250 – 255, blue: 250 – 255), using NIS-Elements AR edgeR. Since the present study aimed to profile genes with
(v4.51.00, Nikon).
a high potential for interaction with AGT, miscellaneous
2.3. Bulk RNA sequencing transcripts such as duplicated, unnamed, ribosomal,
and mitochondrial genes were removed. Criteria for
Liver samples were snap-frozen in a homogenization removal was based on searches within Ensembl gene
solution (#Z305H, Promega, USA) containing identifiers for “NA,” duplicated identifiers, or identifiers
1-thioglycerol (#A208B, Promega). Subsequently, containing: “Gm[0 – 9],” “[0 – 9]Rik,” “RP[2 – 9],” or “mt-
messenger RNA (mRNA) was extracted using Maxwell .” Subsequently, p-values were adjusted using the false
RSC simplyRNA Tissue Kits (#AS1340, Promega) discovery rate (FDR) method. FDR-adjusted p<0.05 was
according to the manufacturer’s protocol. Samples were considered statistically significant. Gene ontology (GO)
processed individually for bulk RNA sequencing, with enrichment analysis of biological processes was performed
single mice representing independent experimental using the clusterProfiler package (v4.2.2) after mapping
replicates. RNA samples were shipped to Novogene Entrez gene identifiers with the org.Mm.eg.db package
(USA) for bulk mRNA sequencing. The sequencing (v3.14.0) on R (v4.1.0). 23-25 Chord plot illustration was
library was generated from total mRNA (1 µg) using performed using the SRplot online tool. 26
NEBNext Ultra RNA Library Prep Kits for Illumina
™
(New England BioLabs, USA). cDNA libraries were then 3. Results
sequenced by a Next Generation NovaSeq platform,
(HWI-ST1276, Illumina, USA), in a pair-end fashion 3.1. Macrovesicular liver steatosis was present after
to reach more than 1,500,000 reads. FASTQ sequence 14 days of WD feeding in hepAGT +/+ mice
data were mapped to the reference mouse genome using We first determined the temporal evolution of WD-induced
STAR (v2.6.1d; https://academic.oup.com/bioinformatics/ liver steatosis in LDLR -/- mice. WD-induced liver steatosis
article/29/1/15/272537?login=false) and quantified using is closely associated with body weight gain, 27,28 and our
FeatureCounts (v1.5.0-p3; https://academic.oup.com/ previous study revealed that WD-induced body weight
bioinformatics/article/30/7/923/232889?login=false). gain begins after 14 days of WD feeding. We therefore
14
Bulk RNA sequencing data (raw FASTQ and aligned data) harvested liver tissues from LDLR -/- mice at selected
are publicly available at the gene expression omnibus intervals of WD feeding, including baseline and day 14
repository (GEO accession number: GSE291082). (Figure 1A). H&E staining did not detect discernable
pathologies in livers at baseline and 5 days of WD
2.4. Statistical analysis feeding. At 14 days of WD feeding, intracellular vacuoles
Data are presented as median and 25 /75 percentiles. were detectable in a substantial portion of hepatocytes,
th
th
Down triangles represent biological replicates. Statistical indicating macrovesicular steatosis. At 42 days of WD
analyses of Oil Red O quantification and individual target feeding, intrahepatic vacuoles of increased size were
differentially expressed genes (DEGs) were performed present abundantly (Figure 1B). Oil Red O staining was
using SigmaPlot (v15.0, SYSTAT Software Inc., USA). then performed to assess neutral lipid accumulation in
Normality and homogeneity of variance were assessed by livers. Neutral lipids were minimally detected in baseline
Shapiro–Wilk and Brown–Forsythe tests, respectively. Data livers. At 5 days of WD feeding, punctate lipid droplets
with confirmed normal distribution and homogeneous were diffusely present within hepatocytes without evident
variance were analyzed by one-way analysis of variance displacement of nuclei. However, we did not detect a
(ANOVA) followed by Holm–Sidak test. Data that failed statistically significant difference in the extent of Oil

Volume 4 Issue 2 (2025) 73 doi: 10.36922/gtm.6027