proximal tubule epithelial cell line hk2  (ATCC)

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Upregulated NNMT sustains angiogenesis in clear cell renal cell carcinoma through activation of the SDF-1/CXCR4–ERK–CCL20 axis

doi: 10.1007/s13402-025-01115-z

Figure Lengend Snippet: Increased expression of NNMT is observed in ccRCC and predicts an unfavorable prognosis for patients. ( A ) Heatmap of DEGs in normal samples and RCC samples. ( B ) Heatmap of methylation levels of methylation-driven genes (MDGs). ( C ) Venn diagram revealed that identification of 11 DEMDGs in RCC. ( D ) The expression levels and relative methylation levels of 11DEMDGs. ( E ) Kaplan-Meier survival curve showed the correlation of NNMT expression with survival rate of ccRCC in TCGA database. ( F ) The relative methylation level of NNMT. ( G ) Analysis of NNMT mRNA expression in TCGA-KIRC and GEO ( GSE66272 , GSE105261 ) datasets. ( H ) Scatterplot showing the fold change in NNMT mRNA levels in ccRCC and corresponding paracarcinoma specimens ( n = 20). ( I ) Western blot of NNMT expression in 9 pairs of ccRCC and corresponding paracarcinoma specimens. ( J ) IHC of NNMT expression in ccRCC and corresponding paracarcinoma specimens. The data are presented as a representative image. Scale bar, 50 μm. K . Correlation between overall survival (OS) and the relative expression level of NNMT in patients with ccRCC. L . Western blot of NNMT expression in the HK2 and RCC cell lines. Data were analyzed using Student’s t-test or Mantel-Cox. Significant results were presented as * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The ccRCC cell lines 769-P, 786-O, OS-RC2, and ACHN, along with the human immortalized proximal tubule epithelial cell line HK2, human umbilical vein endothelial cells (HUVEC), and HEK293T cells, were sourced from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Methylation, Western Blot

Journal: mBio

Article Title: SARS-CoV-2 ORF3a induces COVID-19-associated kidney injury through HMGB1-mediated cytokine production

doi: 10.1128/mbio.02308-24

Figure Lengend Snippet: ORF3a independently induces renal cell-specific injuries and cell death through activation of NF-kB-mediated cytokine production. ( A) Distinctive subcellular localization of WT and Omicron T223I mutant ORF3a proteins in HK2 cells. The WT protein is predominantly localized on the lysosomes as indicated by anti-LAMP-1 antibody and as we reported previously ( , ). The T223I mutant mostly localizes in the ER and Golgi complex that are indicated by anti-calnexin and anti-giantin antibodies, respectively. ( B) Time-course expression of WT and Omicron ORF3a T223I mutant shows concurrent elevation of KIM-1 protein levels in renal epithelial 293T cells ( A ). 293T cells were transfected with a WT or T223I ORF3a -carrying pCAG plasmid. Transfected cells were collected over time and as indicated in hours of post-transfection (hpt). Numeric numbers 1, 2, and 3 on the top of the Western blot represent vector-only control, WT, and T223I mutant ORF3a, respectively. ( C) Expression of WT and T223I mutant proteins triggers NF-kB-mediated cytokine production. ( a) Immunostaining of a RelA/p65 subunit of NF-kB in ORF3a-expression HK2 cells shows that nuclear entry of p65 is specifically associated with ORF3a, an indication of ORF3a-mediated NF-kB activation. Cells were examined at 24 hpt. ( b) ORF3a induces NF-kB activation as measured by an NF-kB promoter firefly luc assay , and upregulation of cytokines TNFα, IL-6, and IFNβ1 by RT-qPCR. ( D) Both WT and T223I ORF3a induce apoptosis and necrosis as measured by a RealTime-Glo apoptosis and necrosis assay (Promega). The levels of all markers were measured by RT-qPCR. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Two human renal cell lines were utilized in this study: the human embryonic epithelial cell line HEK293T (ATCC CRL-1573) and the human renal proximal tubule epithelial cell line HK2 (ATCC CRL-2190).

Techniques: Activation Assay, Mutagenesis, Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Immunostaining, Quantitative RT-PCR

Journal: mBio

Article Title: SARS-CoV-2 ORF3a induces COVID-19-associated kidney injury through HMGB1-mediated cytokine production

doi: 10.1128/mbio.02308-24

Figure Lengend Snippet: Glycyrrhizin (GL4419) blocks viral replication and mitigates ORF3a-induced renal cell damage through HMGB1. ( A) The two chemical structures shown below glycyrrhizin are examples of glycyrrhizin derivatives we tested, including enoxolone (NSC-35347) and a dimer of glucuronic acid (NSC-2559). A list of glycyrrhizin-related compounds is listed in . (B) Treatment of GL4419 suppresses T223I ORF3a-induced cell death, displaying minimal cytotoxicity with a selectivity index of 22.11. ( C) ORF3a mitigates ORF3a-induced apoptosis and necrosis. Tests were carried out in 293T cells at 24 hpt. Cell viability was measured by MTT, cell death by trypan blue, and apoptosis and necrosis by a RealTime-Glo Apoptosis and necrosis assay (Promega). The selectivity index was calculated by the ratio of EC 50 over CC 50 based on the MTT assay. (D-a). GL4419 inhibits viral replication, with an IC 50 of 50.05 ± 5.22 µM, assessed in a stable BHK21 cell-based SARS-CoV-2 NanoLuc replicon system using a Nano-Glo Luciferase Assay (Promega). (D-b) . GL4419 inhibits SARS-CoV-2 replication in VeroE6 cells, exhibiting an EC 50 of 271.8 ± 54.57. The WT USA-WA1/2020 virus was used for infection with titer of 100 TCID 50 , and the cells were collected at 48 hpi. ( E) Treatment of ORF3a-expressing 293T cells with GL4419 (300 µM) reduces KIM-1 protein production possibly through protein degradation of ORF3a. 293T cells transfected with WT and T223I ORF3a were collected at 24 hpt. GL4419 dissolved in DMSO in the concentration of 300 µM was used. DMSO only was used as a negative control. western blotting results are shown in panel a with quantification shown in panel b . Results of RT-qPCR of transfected cells were collected at 48 hpt ( c ). (F) treatment of WT and T223I ORF3a-expressing HK2 cells with GL4419 block activation of NF-kB (indicated by the RelA/p65 subunit of NF-kB) and HMGB1. Expression of ORF3a without GL4419 treatment (mock with DMSO added) activates HMGB1 ( a ) and NF-kB (b) by promoting contrasting nuclear entry and nuclear exit, respectively. In contrast, adding GL4419 (300 µM) results in the retainment of HMGB1 in the nucleus ( a ) and NF-kB in the cytoplasm ( b ).

Article Snippet: Two human renal cell lines were utilized in this study: the human embryonic epithelial cell line HEK293T (ATCC CRL-1573) and the human renal proximal tubule epithelial cell line HK2 (ATCC CRL-2190).

Techniques: MTT Assay, Luciferase, Virus, Infection, Expressing, Transfection, Concentration Assay, Negative Control, Western Blot, Quantitative RT-PCR, Blocking Assay, Activation Assay