Journal: International journal of oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Figure 1. Representative images of the grading system used in the tissue microarray analysis. (A‑E) Different staining intensities of PARK2 in (A) non‑malig‑ nant and (B‑E) ccRCC tissue are shown; (B) negative, (C) weakly positive, (D) moderately positive, (E) strongly positive. (F‑I) Different staining intensities of CKS2 in (F) non‑malignant (G‑I) and ccRCC tissue (G) weakly positive, (H) moderately positive, (I) strongly positive.

Article Snippet: PARK2 and PARK2‐C431S lentiviral plas‐ mids were generated by sub‐cloning the human wild‐type (from plasmid pRK5‐HA‐Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP‐parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX‐EF1α‐IRES‐mCherry (Takara Bio USA, Inc.).

Techniques: Microarray, Staining

Journal: International journal of oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Figure 2. PARK2 mRNA is downregulated in ccRCC tissues and primary cells compared with non‑malignant tissue. (A) PARK2 mRNA expression was exam‑ ined by RT‑qPCR, which showed lower PARK2 expression in tumor tissue of patients with ccRCC compared with non‑malignant tissue. Data were analyzed using Wilcoxon signed‑rank test; n=63; ****P<0.0001. (B) PARK2 mRNA expression levels in ccRCC tissue and primary cells compared with non‑malignant tissue was measured by RT‑qPCR. ccRCC tissue showed decreased PARK2 mRNA expression levels compared with non‑malignant tissue, which were reduced even more when cells were cultivated. Data are presented as the mean ± SEM; n=7. ccRCC, clear cell renal cell carcinoma; PARK2, parkin; RT‑qPCR, reverse transcription‑quantitative PCR.

Article Snippet: PARK2 and PARK2‐C431S lentiviral plas‐ mids were generated by sub‐cloning the human wild‐type (from plasmid pRK5‐HA‐Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP‐parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX‐EF1α‐IRES‐mCherry (Takara Bio USA, Inc.).

Techniques: Expressing

Journal: International journal of oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Figure 3. Generation and functional analyses of cell lines. (A) Transduction of ccRCC cell lines with a lentiviral vector with or without PARK2. (B) PARK2 mRNA expression was examined by reverse transcription‑quantitative PCR, which showed higher mRNA expression levels in PARK2‑transduced cells. TATA‑box binding protein and β‑actin were used as housekeeping genes. Kruskal‑Wallis test was performed followed by Dunn's post hoc test; *P<0.05. (C) PARK2 protein expression levels were assessed by immunoblotting of the different transduced cell lines. β‑actin was used as a loading control. Adjusted densities were measured in GraphPad Prism version 8.2.0. (D) Transwell Boyden chamber migration assays were performed, which demonstrated a difference in migrative capacity after 6‑8 h incubation. Magnification, x10. Data were compared by Kruskal‑Wallis followed by Dun's; n=3; ****P<0.0001. (E) PARK2 overexpression reduced the invasiveness compared with control cells after 48 h incubation. Magnification, x10. Kruskal‑Wallis test was performed followed by Dunn's post hoc test; n=3; ***P<0.001, ****P<0.0001. Data are presented as the mean ± SEM of triplicate experiments. EF1a, elongation factor 1α; EV, empty vector; IRES, internal ribosomal entry site; PARK2, parkin; RT‑qPCR, reverse transcription‑quantitative PCR; WT, wild‑type.

Article Snippet: PARK2 and PARK2‐C431S lentiviral plas‐ mids were generated by sub‐cloning the human wild‐type (from plasmid pRK5‐HA‐Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP‐parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX‐EF1α‐IRES‐mCherry (Takara Bio USA, Inc.).

Techniques: Functional Assay, Transduction, Plasmid Preparation, Expressing, Binding Assay, Western Blot, Control, Migration, Incubation, Over Expression

Journal: International journal of oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Figure 4. CKS2 protein levels are reduced in cells overexpressing PARK2. (A) Volcano plots depicting differential protein expression in the different trans‑ duced cells. Only slight differences in protein levels were observed for 786‑OWT and 786‑OEV cells (|log2FC|>1; count, 4), bigger differences were observed when comparing 786‑O [|log2FC|>1; count, 43 (2 with FDR <0.05)] or 786‑OEV cells [|log2FC|>1; count, 38 (2 with FDR <0.05)] with 786‑OPARK2 cells. (B) Profile plots illustrated that the abundance of PARK2 peptides were increased in 786‑OPARK2 cells. (C) Abundance of CKS2 peptides was decreased in 786‑OPARK2 cells. CKS2, CDC28 protein kinase regulatory subunit 2; EV, empty vector; FC, fold change; PARK2, parkin; WT, wild‑type.

Article Snippet: PARK2 and PARK2‐C431S lentiviral plas‐ mids were generated by sub‐cloning the human wild‐type (from plasmid pRK5‐HA‐Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP‐parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX‐EF1α‐IRES‐mCherry (Takara Bio USA, Inc.).

Techniques: Expressing, Plasmid Preparation

Journal: International journal of oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Figure 5. An inactive catalytic domain mutant 786‑OC431S exhibits a similar phenotype as the control cells. (A) Cells with a PARK2 C431S mutation were gener‑ ated by transducing cells with the pLVX‑EF1a‑PARKC431S‑IRES‑mCherry plasmid. (B) PARK2 mRNA expression levels were examined by RT‑qPCR. TBP and β‑actin were used as housekeeping genes. Data are presented as the mean + SEM of triplicate experiments. Kruskal‑Wallis test was performed followed by Dunn's post hoc test; *P<0.05. (C) RT‑qPCR analysis revealed no difference in CKS2 mRNA expression levels between the different transduced ccRCC cell lines. Data are presented as the mean ± SEM. (D) Transwell Boyden chamber migration assays were conducted with mutant cell lines 786‑OC431S and RCC‑MHC431S. Magnification, x10. Data were compared by Kruskal‑Wallis followed by Dunn's post hoc test; n=3; *P<0.05, ***P<0.001, ****P<0.0001. (E) CKS2 mRNA expression levels were assessed after siRNA knockdown. The experiment was conducted in triplicate. TBP and β‑actin served as housekeeping genes. Data are presented at the mean ± SEM. (F) Migratory capacity was assessed after siRNA‑mediated knockdown of CKS2 and (G) significance was assessed by Kruskal‑Wallis followed by Dunn's post hoc test; n=2; **P<0.01, ***P<0.001. Magnification, x10. ccRCC, clear cell renal cell carcinoma; CKS2, CDC28 protein kinase regulatory subunit 2; EF1a, elongation factor 1α; EV, empty vector; IBR, in‑between RING fingers domain; IRES, internal ribosome entry site; ns, not significant; PARK2, parkin; RT‑qPCR, reverse transcription‑quantitative PCR; siRNA, small interfering RNA; TBP, TATA‑box binding protein; Ubl, ubiquitin‑like domain; WT, wild‑type.

Article Snippet: PARK2 and PARK2‐C431S lentiviral plas‐ mids were generated by sub‐cloning the human wild‐type (from plasmid pRK5‐HA‐Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP‐parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX‐EF1α‐IRES‐mCherry (Takara Bio USA, Inc.).

Techniques: Mutagenesis, Control, Plasmid Preparation, Expressing, Migration, Knockdown, Small Interfering RNA, Binding Assay

Journal: International journal of oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Figure 6. Elevated CKS2 levels are associated with poor survival in patients with ccRCC. (A) Kaplan‑Meier curves of the survival probability showing an improved overall survival for patients with low CKS2 expression levels compared with high CKS2 levels. Significance was assessed with log‑rank test. Number at risk indicates the number of patients which are still alive at given time point; n=247. (B) Kaplan‑Meier curves followed by log‑rank test showing no significant difference in overall survival for patients with high PARK2 expression levels compared with low PARK2 levels; n=250. (C) Cox proportional hazard ratio showing an association between survival and different parameters, such as CKS2 expression, PARK2 expression, tumor grade and pT. *P<0.05, ***P<0.001 (D) Pearson's correlation analysis depicts a relationship between PARK2 and pT. In addition, CKS2 and pT were also analyzed. Bold values indicate significant p‑values. (E) Spearman's correlation of CKS2 and grading as well as PARK2 and grading are depicted. (F) Pearson's correlation between CKS2 and PARK2 including Pearson's r, P‑value, 95% CI, t‑stat and df. ccRCC, clear cell renal cell carcinoma; CI, confidence interval; CKS2, CDC28 protein kinase regulatory subunit 2; df, degrees of freedom; PARK2, parkin; pT, pathologic tumor stage; t‑stat, t‑statistic.

Article Snippet: PARK2 and PARK2‐C431S lentiviral plas‐ mids were generated by sub‐cloning the human wild‐type (from plasmid pRK5‐HA‐Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP‐parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX‐EF1α‐IRES‐mCherry (Takara Bio USA, Inc.).

Techniques: Expressing