Page 87 - JCTR-11-5

Journal of Clinical and
Translational Research ROCK inhibition in chronic rejection

USA) at 50 ng/mL, at 37°C and 5% carbon dioxide. phenylmethanesulfonyl fluoride (#8553, Cell Signaling,
A total of 2 × 10 cells were seeded in each T75 flask. After USA), and prepared for Western blotting as described
7
24 h, the medium was changed to remove non-adherent previously. Western blot bands were normalized to
10
cells, leaving only monocytes (adherent cells). Cells were glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
differentiated into macrophages (human monocyte- which was used as a loading control (protein of interest
derived macrophages [HMDM]) over 6 days. On day 6, the vs. GAPDH), and each experiment was repeated three
HMDM were treated with inhibitors. times to reduce technical noise. Statistical analysis was
performed to ensure data significance.
2.5. Treatment with inhibitors
At 70% confluency, cells were treated for 24 h with 2.9. Antibodies
Rezurock (HY-15307, MedChem Express, United States of The following primary antibodies were used:
America [USA]) at a concentration of 10 µM, fingolimod ROCK2 (8236s, Cell Signaling, USA), Notch1 (3608s,
(S5002, Selleckchem, USA) at 300 nM, or Rezurock and Cell Signaling, USA), pentraxin 3 (PTX3; PA5-36156,
fingolimod in combination. For HMDM, Rezurock Invitrogen, China), collagen Type1 (14695-1-AP,
alone or in combination with fingolimod was used at a Proteintech, USA), chemokine (C-C motif) ligand 2 (CCL2;
concentration of 5 µM. Dimethyl sulfoxide (DMSO; Sigma, MA5-17040, Invitrogen, China), C-C motif chemokine
USA) was used as a control, and its volume matched the receptor 2 (CCR2; MA5-42780, Invitrogen, China), and
volume used for the highest drug concentration to ensure transforming growth factor beta 1 (TGF-β1; 21898-1-
consistent DMSO levels across all samples. AP, Proteintech, USA), all at 1:1,000 dilution. ROCK1
2.6. RNA isolation antibody (ab199899, Abcam, USA; currently discontinued;
https://www.abcam.com/en-us/products/unavailable/
Mouse peritoneal macrophages were pelleted and sent rock1-antibody-c-terminal-ab199899) was used at 1:2,000
to Active Motif, Inc. (USA) for RNA isolation, library dilution, and GAPDH (14C10, Cell Signaling, USA) at
preparation, and sequencing analysis. 1:3,000 dilution. For secondary antibodies, anti-rabbit

2.7. RNA sequencing and analysis immunoglobulin G (IgG; 7074P2, Cell Signaling, USA)
and anti-mouse IgG (7076, Cell Signaling, USA) were used
Next-generation sequencing was performed using the at 1:5,000 dilutions.
Illumina platform, and Venn diagrams were generated as
described previously. Genes selected for the Metascape 2.10. Statistics
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Gene Ontology (GO) analysis had a p≤0.05 and log fold For western blotting (protein expression) analysis, three
2
change‖ > 0. All genes considered differentially expressed biological replicates were used to ensure statistical power,
in the treated samples, compared to the control, had a as described previously. Differentially expressed genes
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p≤0.05 and ‖log fold change‖ > 0. For RNA sequencing (DEGs) were selected using a p-value cutoff of < 0.05, and
2
(RNA-seq) analysis, two biological replicates were used for a log fold-change > 0. These thresholds were applied when
each treatment condition. In our gene expression analysis, generating volcano plots for each treatment group.
2
sequencing quality was ensured by filtering raw reads to
obtain clean reads. This was followed by alignment to the The protein expression changes after drug treatment
reference genome using HISAT2 and STAR tools. Gene were consistent with the transcriptomic findings,
expression levels were normalized using the “Fragments Per supporting the reliability of the DEG selection. Pathway
Kilobase of exon per Million mapped reads” method, and enrichment and GO analyses were carried out using the
principal component analysis plots were used to visualize Metascape platform based on DEGs with p≤0.05 and a log
2
sample clustering and assess potential batch effects. All fold change > 0. Results were interpreted using bar plots
samples were processed under identical conditions to and clustering analyses to identify significantly enriched
minimize technical variability. biological processes. RNA-seq data were analyzed using
DESeq2, which includes internal normalization and applies
2.8. Cell lysis and western blots the Wald test followed by Benjamini–Hochberg correction

Control, Rezurock-, fingolimod-, and Rezurock/ to control the false discovery rate (FDR). All pathway
fingolimod-treated RAW 264.7 and HMDM macrophages enrichment analyses (e.g., GO and Kyoto Encyclopedia of
were lysed in radioimmunoprecipitation assay cell lysis Genes and Genomes [KEGG]) were likewise corrected for
buffer (#9806, Cell Signaling, USA) supplemented with 1× multiple hypothesis testing using FDR-adjusted p-values.
protease inhibitor (cOmplete™, Mini, EDTA-free Protease Statistical significance thresholds (adjusted p<0.05) were
Inhibitor Cocktail, #11836170001, Roche, USA) and 1× applied consistently across all analyses.

Volume 11 Issue 5 (2025) 81 doi: 10.36922/JCTR025270036