Fig 1: Differentially expressed genes (DEGs) after hepatocyte nuclear factor 4α (HNF4A) transduction of primary proximal tubular epithelial cells are associated with the proximal tubular phenotype. A: Volcano plot of RNA-sequencing (RNA-seq) data reveals 2033 significant DEGs in HNF4A-transduced cells compared with Null (blue and red). After further filtering, 130 genes remained [adjusted P (Padj) < 10−8, |log2 fold change| > 2; marked by dashed black line and gene transcript positions in red]. Of these, eight genes (AGMAT, ANKS4B, CDHR2, CDHR5, HPD, LRP2, MYO7B, and RARRES1) were selected for further confirmation based on their role as known HNF4A target genes, function, and histologic expression pattern. B: Quantitative PCR of HNF4A and the selected genes demonstrates significant up-regulation at the RNA level in all cases 4 days after HNF4A transduction. Statistical significance is determined by paired t-test. C: Western blot analysis shows up-regulation at the protein level of HNF4A, ankyrin repeat and sterile α motif domain-containing 4B (ANKS4B), agmatinase (AGMAT), 4-hydroxyphenylpyruvate dioxygenase (HPD), retinoic acid receptor responder 1 (RARRES1), low-density lipoprotein receptor-related protein 2 (LRP2), and cadherin-related family member 2 (CDHR2) 8 days after HNF4A transduction. D: RNA-seq data analysis displays modest alterations and no systematic pattern of expression of established scattered tubular cell markers [A-kinase anchoring protein 12 (AKAP12), B-cell lymphoma 2 (BCL2), caveolin 1 (CAV1), CD24, keratin 7 (KRT7), polymeric Ig receptor (PIGR), prominin 1 (PROM1), SRY box transcription factor 9 (SOX9), vascular cell adhesion molecule 1 (VCAM1), and vimentin (VIM)] following HNF4A transduction. Data are presented as means ± SEM (B and D). n = 3 (B); n = 4 (D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (B); ∗Padj < 0.05, ∗∗∗∗Padj < 0.0001 (D). CDHR5, cadherin-related family member 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MYO7B, myosin VIIB.
Fig 2: Multi-omics characterization identified RARRES1 as a core mediator of chemotherapy resistance in the subcluster c05. A UMAP plot showed the number of differentially expressed genes (DEGs) between PD and PR/SD samples across malignant epithelial subclusters. B Volcano plot of DEGs from c05 versus other malignant subclusters. C Intersection of DEGs from four comparisons: (i) c05 vs. other malignant subclusters, (ii) PD vs. Naive/PR/SD, (iii) metastasis vs. primary tumors, and (iv) chemotherapy-exposed vs. naive samples, identifies consistent upregulation of SNCG, CST6, CAPS, and RARRES1. D Transcriptome profile confirmed significantly elevated RARRES1 expression in L3.6pl-Res compared to L3.6pl wild type. E Western blot showed increased RARRES1 protein abundance in L3.6pl-Res cells compared to sensitive L3.6pl wild type cells. (F, G) L3.6pl-Res cells were either stably transduced using Lentiviral-particles with two different shRNAs (A + B) targeting human RARRES1 or with a non-targeting sh-Control. RNA and protein-lysates were extracted. Real-time RT-PCR was conducted to quantify and compare RARRES1 mRNA-expression in L3.6pl-Res cells transduced with shRNAs (A + B) or sh-Control (F). RNAi-mediated effective depletion of RARRES1 protein-levels, compared to sh-Control. Actin was used as loading control (G). **, P < 0.01; *, P < 0.05. H RARRES1 knockdown L3.6pl-Res cells (A + B) and sh-Control cells were treated with increasing gemcitabine concentrations for 48 h, or left untreated (0 nM) and cell viability was measured by MTT assay. L3.6pl-Res wildtype (non-transduced) cells were used as additional control. RNAi-mediated RARRES1 knockdown (A + B) is linked to increased gemcitabine chemosensitivity, compared to sh-Control cells. The half maximal inhibitory concentration (IC50) of gemcitabine is depicted (dotted line). I Pseudotime trajectory analysis showed progressive enrichment of RARRES1 expression at terminal trajectory stages associated with chemotherapy resistance. J, K TIDE analysis revealed that high-RARRES1 groups exhibited comparable immune dysfunction scores but significantly elevated immune exclusion scores compared to low-RARRES1 groups. L Scatter plot showed a positive correlation between the RARRES1 gene expression and exclusion scores. M Pan-cancer survival analysis established RARRES1 overexpression as a prognostic biomarker for reduced overall survival across multiple malignancies
Supplier Page from Abcam for Anti-RARRES1 antibody