PMC

Characterization of rat fibroblast cultures. (A) The fibroblasts exit from tissue fragments reviewed under a microscope on the 4^th day in culture. (B) Typical spindle morphology of the fibroblasts evaluated after HE staining. All primary culture cells at the 25^th passage were confirmed to be positive for Vimentin by immunofluorescence microscopy (C), but occasionally some cells were positive for α-SMA as well (D).

Metabolic reprogramming of the FH^+/– cells. (A) Significantly more cellular fumarate production, and the bar graphs represent the fumarate concentration detected with the assay kit. (B) Extracellular lactate excretion and cytoplasmic lactate production. (C) Representative glucose consumption results examined by flow cytometry, and the corresponding histogram is shown in (D). (E) Representative OCR results investigated by a Seahorse XFe96 Extracellular Flux Analyser and (F) the OCR histograms. A representative flow cytometry of ROS is shown in (G), and the corresponding histogram is shown in (H), where the bar graphs show the percentages of ROS⁺ cells among the WT and FH^+/– cells.

Results of transcriptome sequencing. (A) Scatter plots of all expressed genes by BGISEQ-500. Blue represents downregulated genes, red represents upregulated genes and brown represents non-regulated genes. (B) and (C) The results of the pathway function analysis of differentially expressed genes. The up and down of differentially expressed genes is shown in (B), where the X-axis indicates pathway entry, the Y-axis represents the number of genes and up and down for the corresponding pathway entry. (C) The results of differential gene pathway enrichment, where the X-axis represents the enrichment factor and the Y-axis represents the pathway name. (D) Genes of the pathways in cancer. The RT-qPCR validation of the selected DEGs revealed by transcriptome sequencing is shown in (E).

Reduced FH gene and protein expression in FH^+/– cells. (A) FH gene sequencing results for WT and two different situations of FH^+/– cells. (B) The 11 base pair deletion in one allele in exon 1 in FH^+/– cells, which is marked in red in the opposite allele. Significantly reduced FH mRNA expression detected by RT-qPCR is shown in (C). Western blot analysis of FH protein expression is shown in (D), and the corresponding histogram is shown in (E), where the bar graphs represent FH protein expression normalized by β-actin. (F) Immunofluorescence microscopy of FH protein expression (200×).

Effect of FH^+/– KO on proliferation, apoptosis and cell cycle. All experiments were performed on the cells at approximately 25 passages. (A) and (B) The growth curves and images, respectively, generated by the Incucyte Zoom system. A representative flow cytometry cell cycle analysis is shown in (C), and the corresponding histogram of the cell cycle analysis is shown in (D), where the bar graphs represent the percentage of WT and FH^+/– cells in the G2/M, S and G0/G1 phases. Early apoptosis detected by flow cytometry is shown in (E), and the corresponding histogram is shown in (F), where the bar graphs represent the percentage of apoptotic cells.

Further characterization of the FH^+/– cells. (A) Microscopic images of WT and FH^+/– cells at the 61^st passage (200×). (B) Representative ultrastructure images of WT and FH^+/– cells at the 55^th passage evaluated by electron microscopy (a,d:2000×; b,e:4000×; c.f:8000×), where a larger cell body with plenty of rough endoplasmic reticulum, mitochondria and ribosomes is shown in the FH^+/– cells, but the WT fibroblasts show fewer mitochondria and ribosomes and deformed and less condensed rough endoplasmic reticulum. (C) Representative immunofluorescence images of the expression of p53, p21 and p16 (200×). Western blot for p53, p21, p16 and TERT in WT and FH^+/– cells and the corresponding histograms are shown in (D) and (E), respectively. (F) SA-β-Gal detection . (G) FH^+/– single cell cloning images captured with the Incucyte Zoom System for cells at the 84^th passage. (H) The karyotype of a RLFHF FH^+/– cell, where no chromosome abnormality is seen as revealed for the karyotype of a control fibroblast at the 40^th passage. (I) Electron microscope images of RLFHF FH^+/– cells (4000× for both a and b), which are rich in mitochondria, rough endoplasmic reticulum and ribosomes. Western blot for FH protein expression in the 58^th passage WT, 68^th passage FH^+/– and RLFHF cells is shown in (J) and corresponding histograms are shown in (K).