Fig 1: DNAJC24 knockdown inhibits HCC cells proliferation and autophagy by affecting the metabolism of ammonia.A Interactions in the STRING protein interaction database were used to analyze DEGs interaction networks. B, C GO functional enrichment analysis (B) and KEGG pathway enrichment analysis (C) of DEGs was performed to identify functionally related gene pathways. D Western blotting was performed to determine protein levels of CPS1. E, F NH4+/NH3 levels were measured in the PLC-KD (E) cells or Huh7-KD (F) cells as well as in the corresponding control cells using an ammonia assay kit by spectrophotometric analysis at 570 nm with extrapolation from the standard curve and expressed in nmol/mg protein. G, H NH4Cl (final concentration 25 mM) or/and Bafilomycin A1 (final concentration 400 nM) was added to the culture medium of PLC or Huh7 and incubated for 24h, respectively. Western blotting was performed to determine protein levels of LC3B. I, J CCK-8 cell viability assay analysis of the impact of NH4Cl (final concentration 25 mM) on PLC (I) and Huh7 (J) cell growth. Results were normalized to viability at day 0 and represented as fold change. Data were presented as mean ± SEM. n = 3–5. **P < 0.01, ****P < 0.0001.
Fig 2: DNAJC24 promotes proliferation and motility of HCC cells in vitro.A, C PLC and Huh7 cells were infected with a lentivirus to produce stable DNAJC24 overexpression (OE) cells. qRT-PCR was performed to determine levels of DNAJC24 mRNA. ß-actin was used as an internal control. B, D Western blotting was performed to determine levels of DNAJC24 protein. E, G CCK-8 cell viability assay analysis of the impact of DNAJC24 ectopic overexpression on PLC (E) and Huh7 (G) cell growth. Results were normalized to viability at day 0 and represented as fold change. F, H The effect of ectopic overexpression of DNAJC24 on the proliferation of PLC (F) and Huh7 (H) cells was analyzed by EdU staining. Representative images and EdU positive cell rates are as shown. I–L Chemotaxis (I, K) and Matrigel invasion (J, L) assays were used to detect the effect of ectopic overexpression of DNAJC24 on PLC (I, J) and Huh7 (K, L) motility. Data were presented as mean ± SEM. n = 2–5. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 3: Knockdown of DNAJC24 inhibits proliferation, motility, and protein synthesis in HCC cells in vitro.A, C PLC and Huh7 cells were infected with a lentivirus to produce stable DNAJC24 knockdown (KD) cells. qRT-PCR was performed to determine levels of DNAJC24 mRNA. ß-actin was used as an internal control. B, D Western blotting was performed to determine levels of DNAJC24 protein. E, H CCK-8 cell viability assay analysis of the impact of DNAJC24 knockdown on PLC (E) and Huh7 (H) cell growth. Results were normalized to viability at day 0 and represented as fold change. F, I Colony formation assay showing the effects of DNAJC24 knockdown on PLC (F) and Huh7 (I) cell growth. G, J Immunofluorescence staining detected Ki67 expression in DNAJC24-KD cells and control cells. Representative images and Ki67 positive cell rates are as shown. K, L, N, O Chemotaxis (K, N) and Matrigel invasion (L, O) assays were used to detect the effect of DNAJC24 knockdown on PLC (K, L) and Huh7 (N, O) motility. M, P Newly synthesized protein was detected in PLC (M) and Huh7 (P) cells after DNAJC24 knockdown using a protein synthesis assay kit. Data were presented as mean ± SEM. n = 2–3. *P < 0.05, **P < 0.01.
Fig 4: External stimuli such as starvation, hypoxia and heat upregulate DNAJC24 expression through HSF2 in HCC cells.A The cell culture medium of PLC and Huh7 was changed to PBS and the cells were cultured in PBS for 0h, 3h and 6h respectively and then the cells were lysed. Western blotting was performed to determine levels of DNAJC24 protein. B Adding CoCl2 (final concentration 200 µM) to the medium of PLC and Huh7 cells to simulate hypoxic environment, and the cells were incubated in it for 0h, 4h, 6h, 8h respectively and then lysed. Western blotting was performed to determine levels of DNAJC24 protein. C PLC and Huh7 cells were cultured at 37 °C, 39 °C, 41 °C, 43 °C respectively in 5% CO2 for 3h, and then the cells were lysed. Western blotting was performed to determine levels of DNAJC24 protein. D–F Analysis of the possible correlation between DNAJC24 and HSF1 (D), HSF2 (E), HSF4 (F) mRNA levels based on the data from the TCGA HCC dataset. Data were analyzed using Spearman’s rank correlation coefficient. G Box plots of HSF2 expression in cancer and paired normal liver tissues of HCC patients based on data from the TCGA HCC dataset. H Box plots of HSF2 expression in normal liver and tumor tissues (Clinical stages I–III) based on data from the TCGA HCC database. I The Kaplan–Meier survival analysis of overall survival of HCC patients analyzed by GEPIA (top 50%, high; bottom 50%, low). J Lysis of PLC and Huh7 cells after applying starvation, hypoxia, and heat (40 °C) stimulation to 3h. Western blotting was performed to determine levels of HSF2 protein. K Stable cell lines with HSF2 overexpression were constructed using lentiviral transfection in PLC and Huh7 cells, and the protein levels of HSF2 and DNAJC24 were detected by Western blotting after cell lysed. L DNAJC24 wild-type promoter (pPRO-RB-promoterWT) and mutant promoter (pPRO-RB-promoterMUT)were ligated to the reporter vector. A dual luciferase assay was performed to assess the effects of HSF2 overexpression on DNAJC24 transcription in PLC cells. Data were presented as mean ± SEM. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 5: DNAJC24 expression is generally upregulated in human HCC tissues and is associated with poor patient prognosis.A Expression of DNAJC24 in cancer and paired normal liver tissues of 50 HCC patients based on data from the TCGA hepatocellular carcinoma dataset. B, C The Kaplan–Meier survival analysis of disease-free survival (B) and overall survival (C) of HCC patients analyzed by GEPIA (top 20%, high; bottom 80%, low). D Representative images of DNAJC24 in 167 HCC and paired para-tumor tissues stained with IHC (scale bar,200 µm or 100 µm). E IHC analysis of DNAJC24 expression in 167 pairs tissue of HCC specimens. F Positive rate of DNAJC24 in different tumor size subgroups (Group A, 25 cases; Group B, 114 cases; Group C 28 cases). G Percentage of high and low microvessel density in the DNAJC24 positive and negative subgroups. H, I The Kaplan–Meier survival analysis of recurrence-free survival (H) and overall survival (I) in 167 HCC patients. J Box plots of DNAJC24 expression in normal liver and tumor tissues (Clinical stages I to III) from the TCGA Database. *P < 0.05, ****P < 0.0001.
Supplier Page from Abcam for Anti-DPH4 antibody