Fig 1: HADH was downregulated in ccRCC clinical specimens and cell lines. (A) Relative HADH mRNA expression in the frozen ccRCC tissues compared with paired normal renal tissues was detected by RT-PCR. β-actin was used as an internal control. (B and C) Western blot of HADH expression in the frozen ccRCC tissues compared with paired normal renal tissues. HSP90 was used as a loading control. (D) Gross tissue microarray images showed IHC staining of HADH in 16 pairs of ccRCC and adjacent normal tissues. (E and F) Representative images of IHC staining from the cohort shown in Fig. 2D, positive staining (DAB) was denoted by the brown color. (G and H) The protein expression level of HADH in four ccRCC cell lines (ACHN, Caki-1, 786–0 and A498) and normal renal tubular epithelial cell line (HK-2). *** p < 0.001, **** p < 0.0001.
Fig 2: Low expression of HADH was associated with poor prognosis in patients with ccRCC. (A) The protein expression level of HADH in different types of malignant tumors between adjacent normal tissues and cancer tissues in CPTAC database. (B) The mRNA expression level of HADH in different types of malignant tumors between adjacent normal tissues and cancer tissues in TCGA database. (C) The mRNA expression level of HADH between ccRCC tissues (n=538) and adjacent normal tissues (n=72) in the TCGA KIRC. (D) Overall survival and (E) disease free survival curve of ccRCC patients with low and high HADH expression in TCGA KIRC. Relative expression levels of HADH at different stages (F) and grades (G) in TCGA KIRC. (H) The mRNA expression level of HADH between ccRCC tissues and paired adjacent normal tissues in the GSE40435 dataset (n=101). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 3: HADH negatively regulated the expression of glutathione synthesis genes and nuclear translocation of NRF2 in ccRCC cells. (A) The mRNA expression of GCLC, GCLM and GSS was detected by RT-PCR in ccRCC cells with HADH overexpressed and ccRCC cells with Vector. (B) The protein level of GCLC, GCLM and GSS was detected by Western blot in ccRCC cells with HADH overexpressed and ccRCC cells with Vector. (C) The expression of NRF2 in ACHN cells with HADH overexpressed or Vector was observed by immunofluorescence staining. (D) Protein levels of NRF2 in cytoplasm and nucleus of ACHN and Caki-1 cells with HADH overexpressed or Vector were detected by Western blot. (E) The expression of NRF2 was observed by immunofluorescence staining in ACHN cells infected with plasmid pcDNA3.1 or pcDNA3.1‑NRF2. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4: Downregulation of enzymes involved in fatty acid oxidation in cmGch1 KO hearts. (a–c) Protein levels of three key fatty acid oxidation enzymes (ACSL, acyl‐coenzyme A synthase (long‐chain); ACAD, acyl‐coenzyme A dehydrogenase; HADH, hydroxylacyl‐coenzyme A dehydrogenase). Brain and liver tissues were used as negative (−ve) and positive (+ve) controls, respectively. (d) Intracellular triacylglycerol (TAG) levels are increased in cmGch1 KO hearts compared to WT. (e) A schematic diagram of fatty acid catabolism pathway. Fatty acid can either undergo oxidation for ATP production or be incorporated into TAG. Fold changes (log) of mRNA expression by RNA sequencing are shown in dotted boxes. Data are presented as means ± SD and analysed using unpaired Student's t‐test. Each data point represents an animal used.
Fig 5: Overexpression of HADH inhibited the malignant behaviors of ccRCC cells in vitro. (A) The mRNA level of HADH in ACHN and Caki-1 cells infected with Vector Lentivirus or HADH overexpressed Lentivirus was detected by RT-PCR. β-actin was used as an internal control. (B) The protein level of HADH in ACHN and Caki-1 cells infected with Vector Lentivirus and HADH overexpressed Lentivirus was detected by Western blot. HSP90 was used as a loading control. (C) Cell proliferation ability was detected by CCK8 assay in ACHN and Caki-1 cells. (D) Expression of Ki67 was detected by immunofluorescence staining. (E) Cell proliferation ability was detected by clone formation assay. (F) Tumor cell migration ability was detected by the wounding healing assay in ACHN and Caki-1 cells. (G) Cell migration and invasion ability was detected by the transwell assay in ACHN and Caki-1 cells. (H) The expression level of apoptosis-related protein cleaved Caspase3 was detected by Western blot in ACHN and Caki-1 cells. HSP90 was used as a loading control. (I) Cell apoptosis was detected by a flow cytometry in ACHN and Caki-1 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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