Longstanding Transcriptional Activation of APOA1 and PON1 in Human Hepatocytes by CRISPR/dCas9 Technology: Transcriptomic Profile and Crosstalk with Endothelial Cells

Figures

• 

  Figure 1. APOA1 and PON1 levels in selected and non-selected transfected HuH-7 cells after CRISPR/dCas9 transfection. APOA1 and PON1 mRNA (a,b) in transfected and non-selected HuH-7 cells. APOA1 mRNA (c), intracellular APOA1 protein (d) and secreted APOA1 level (e) in HuH-7 cells transfected with plasmids for APOA1 activation and selected with antibiotic mix, at 46 days after the transfection. PON1 mRNA (f), intracellular PON1 protein (g) and secreted PON1 level (h) in HuH-7 cells transfected with plasmids for PON1 activation and selected with an antibiotic mix, at 46 days after the transfection. The intracellular protein level is expressed relative to β-actin, and the secreted proteins in the culture medium are normalized to the total cell protein. All data are expressed as fold change versus cells transfected with control plasmids (CP) and presented as mean ± SD. *** p < 0.001 versus CP.

• 

  Figure 2. Distribution of differentially expressed (DE) genes identified by RNAseq analysis in transfected and selected hepatocytes for APOA1 or PON1. (a) Heatmap diagram with functional group connections for DE genes distribution in individual samples of transfected and selected HuH-7 cells: red—upregulated genes, green—down-regulated genes; (b) The number of upregulated (gray) and down-regulated (blue) DE genes identified in HuH-7 cells with transcriptional activated APOA1 and PON1, normalized to HuH-7 cells transfected with control plasmid (CP). The statistical thresholds applied to identify the DE genes were edgeR adj.p value ≤ 0.05 and |log2FoldChange| ≥ 0.0; (c,d) Volcano plots expressing DE gene identified in selected HuH-7 hepatocytes with upregulated APOA1 (c) or upregulated PON1 (d) normalized to HuH-7 cells transfected with control plasmid, (CP). The statistical thresholds applied to identify the DE genes were p value ≤ 0.05 and |log2FoldChange| ≥ 0. Red—upregulated genes, green—down-regulated genes, blue—unchanged genes.

• 

  Figure 3. Functional enrichment analysis for DE genes in HuH-7 cells with upregulated APOA1 (a) and PON1 (b) using DAVID tools and Gene Ontology (GO) database. Dot plots illustrate top 10 biological processes (BP, top), molecular functions (MF, middle) and cellular components (CC, bottom) associated with up- and down-regulated DE genes identified using the cutoff thresholds adj.p-value ≤ 0.05 and |log2FoldChange| ≥ 0. Dot size depicts the identified gene count in the functional enrichment group, the dot color represents the adj.p-values of predicted functional enrichment, and GeneRate is the ratio of the identified genes in the functional group to total number of the DE genes.

• 

  Figure 4. Differentially expressed genes in HuH-7 cells with upregulated APOA1 or PON1. Differentially expressed (DE) genes from GO biological process (BP) showing the response to oxidative stress (GO:0034599) in selected HuH-7 cells with transcriptional activated APOA1 (a) or PON1 (b).

• 

  Figure 5. Partial validation of RNAseq results by Western blotting. Secreted PON1 in the conditioned media from APOA1-transfected and selected HuH-7 (a); secreted ALB level in the conditioned media from APOA1 (b) or PON1 (c) transfected and selected HuH-7 cells. Data are expressed as fold change versus cells transfected with control plasmids (CP) and presented as mean ± SD. ** p < 0.01, *** p < 0.001 versus CP.

• 

  Figure 6. Oxidative stress markers in TNFα-activated HUVECs exposed to conditioned media from HuH-7 cells transfected with APOA1 or PON1 plasmids. ROS levels expressed relative to cellular protein (a) and mitochondrial ROS levels expressed relative to cellular protein (b) in TNFα-activated HUVECs. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 versus CP.

• 

  Figure 7. Inflammatory stress markers in TNFα-activated HUVECs exposed to conditioned media from HuH-7 cells transfected with APOA1 or PON1 plasmids. VCAM-1 mRNA (a) and protein (b), and MCP-1 mRNA (c) and protein (d) in TNFα-activated HUVECs exposed to CM from transfected HuH-7. The protein level is expressed relative to β-actin. All data are expressed as fold change versus cells transfected with control plasmids (CP) and presented as mean ± SD. ** p < 0.01, *** p < 0.001 versus CP.

• 

  Scheme 1. Graphical representation of the experimental design. Created in BioRender. Licensed to Niculescu, L. S. (2026) https://BioRender.com/6nxlu8o.