human renal proximal tubular epithelial cells hrptecs  (ATCC)

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Inhibition of BKPyV replication by IFNα HRPTECs were infected with BKPyV (1 × 10 6 copies/mL) for 2 h and then incubated in the presence or absence of human IFNα (0−20 ng/mL) for an additional 72 h. At the end of experiments, cell lysates were collected for Western blot analysis (A) to determine the protein expression of MxA and VP1, and the BKPyV viral titer was measured by qPCR (B) from the collected supernatant. (C) Eight-to ten-week Balb/c female mice received 30 min-ischemic reperfusion injury (IRI) of a unilateral kidney. Mice were then intraperitoneally inoculated with MuPyV wild-type strain (2 × 10 6 plaque-forming units). Two doses of IFNα (1 MIU/Kg) or PBS were subcutaneously injected on day 1 and day 4. After seven days of inoculation, the mice were sacrificed, and fold change of MuPyV VP1 mRNA relative expression using 18S rRNA (18S) as reference in the IRI and non-IRI kidneys was determined by RT-qPCR. The results of cell culture experiments represent three independent experiments. For animal study, each experimental group comprised 4 to 6 animals. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Infection, Incubation, Western Blot, Expressing, Injection, Quantitative RT-PCR, Cell Culture

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Inhibition of BKPyV replication by MxA HRPTECs were transfected with varying doses (0.1a2 μg) of FLAG-tagged MxA-expressing plasmids or an empty plasmid for 12 h. Subsequently, they were infected with BKPyV (1 × 10 6 copies/mL) for 2 h. After replacing the medium, the cells were cultured for an additional 72 h. BKPyV-infected cells transfected with the empty vector served as the negative control, while BKPyV-infected cells treated with 10 ng/mL of IFNα served as the positive control. At the end of the experiments, viral titer analysis (A) was performed on the collected supernatant, and Western blot analysis (B) was conducted on cell lysates to determine the expressions of TAg, VP1, and MxA proteins. Bar graphs depict the normalized levels of BKPyV TAg (C) and VP1 (D) relative to GAPDH. The data represent three independent experiments. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗∗, p < 0.01; ∗∗∗, p < 0.005; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Infection, Cell Culture, Negative Control, Positive Control, Western Blot

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: MxA silencing diminished IFNα′s anti-BKPyV effect HRPTECs were transfected with varying doses of MxA siRNA or scrambled RNA for 12 h. Afterward, they were treated with IFNα (10 ng/mL) and infected with BKPyV (1 × 10 6 copies/mL) for an additional 72 h. Western blot analysis was performed to assess the expression levels of TAg, VP1, and MxA proteins (A). Bar graphs depict the normalized levels of BKPyV MxA (B) and VP1 (C) relative to GAPDH. The data represent three independent experiments. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Infection, Western Blot, Expressing

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Mutation of MxA residue 103 decreases antiviral activity against BK polyomavirus . HRPTECs were transfected with plasmids encoding FLAG-tagged wild-type MxA, GTP binding-defective MxA mutants (K83A and D250 N), or the GTPase-deficient MxA mutant (T103A) for 12 h. Cells transfected with the empty vector served as the negative control. Subsequently, the cells were infected with BKPyV (1 × 10 6 copies/mL) for an additional 72 h. Western blot analysis was conducted to determine the expression levels of wild-type MxA, MxA mutants, BKPyV TAg, and VP1 proteins. Bar graphs represent the normalized levels of BKPyV TAg (B) and VP1 (C) relative to GAPDH. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Residue, Activity Assay, Transfection, Binding Assay, Plasmid Preparation, Negative Control, Infection, Western Blot, Expressing

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: BKPyV TAg is co-immunoprecipitated with wild-type MxA but not with the MxA T103A mutant . A. HRPTECs were co-transfected with BKPyV TAg-expressing plasmid and FLAG-tagged wild-type MxA-expressing plasmid. Control transfections were the TAg-expressing or wild-type MxA-expressing plasmid alone. Immunoprecipitation assay was performed by immunoprecipitation with anti-SV40 TAg antibody. The TAg-binding MxA in the TAg-associated immunocomplexes was assessed by subsequent immunoblotting with an anti-FLAG antibody. The protein levels of inputted MxA, TAg, and GAPDH were determined through immunoblotting using their respective specific antibodies. B. HRPTECs were co-transfected with either FLAG-tagged MxA or FLAG-tagged MxA mutants (K83A and T103A)-expressing plasmids, along with the TAg-expressing plasmid. The immunoprecipitation assay was conducted using an anti-SV40 TAg antibody, followed by immunoblotting with an anti-FLAG antibody.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Control, Binding Assay, Western Blot

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: MxA and BKPyV TAg are colocalized in the cytoplasm . A. HRPTECs were infected with BKPyV (1 × 10 6 copies/mL). After 72 h, cells were fixed and stained for MxA and TAg. B&C. HRPTECs were initially infected with BKPyV (1 × 10 6 copies/mL) for 2 h, followed by incubation in the absence (B) or presence (C) of human IFNα (10 ng/mL) for an additional 72 h. D. HRPTECs were transfected with wild-type MxA-expressing plasmids for 12 h and infected with BKPyV (1 × 10 6 copies/mL). After 72 h, cells were fixed and stained for MxA and TAg. MxA was labeled with anti-MxA and a green fluorescent (Alexa Fluor 488) secondary antibody, and BKV TAg was labeled with anti-SV40 TAg and a red fluorescent (Alexa Fluor 647) secondary antibody. In MxA-expressing cells, MxA exhibited colocalization with BKPyV TAg in the cytoplasm, displaying a diffuse granulate pattern (indicated by white arrowheads) (C&D). In BKPyV infected non-MxA expressing cells, TAg could be seen exclusively in the nucleus (indicated by grey arrows) (A, C&D).

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Infection, Staining, Incubation, Transfection, Expressing, Labeling

Journal: Virus research

Article Title: The essential role of the ERK activation in large T antigen of BK polyomavirus regulated cell migration.

doi: 10.1016/j.virusres.2023.199220

Figure Lengend Snippet: Fig. 3. ERK inhibition effectively suppresses TAg- and tAg-induced cell motility. Stable TAg- or tAg-expressing and LacZ-expressing cells hRPTEC cells were cultured in the presence of either the solvent control (DMSO) or U0126 (10 μM) for 24 h. Expressions of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/ 2) were assessed by Western blot analysis (a). Stable TAg- or tAg-expressing and LacZ- expressing hRPTECs cells were seeded into the inner chamber of the permeable wells. Trans well migration assays were performed as described in the Methods. The number of cells migrating to the outer chamber was photo graphed (b) and quantified (c). The presented data were obtained from three independent experiments. ns indicates; no significance. * indicates p < 0.05. ** indicates p < 0.005.

Article Snippet: Human renal proximal tubular epithelial cells (hRPTECs) cell line (ATCC® CRL-4031TM) was cultured in DMEM/F-12 1:1 (GibcoTM 11330032) supplemented with 1% fetal bovine serum (GibcoTM 10437028) and the hTERT immortalized RPTEC growth kit (ATCC® ACS-4007TM).

Techniques: Inhibition, Expressing, Cell Culture, Solvent, Control, Western Blot, Migration

Journal: Virus research

Article Title: The essential role of the ERK activation in large T antigen of BK polyomavirus regulated cell migration.

doi: 10.1016/j.virusres.2023.199220

Figure Lengend Snippet: Fig. 5. The expression of MMP-2 and MMP-9 is regulated by TAg through ERK signaling. Stable tAg- or TAg-expressing and LacZ-expressing hRPTECs cells were cultured in the presence of either the solvent control (DMSO) or U0126 (10 μM) for 24 h. Culture medium was subsequently collected. The activity of MMP-2 and MMP-9 in the culture medium was assessed by gelatin zymography (a). The quantification of MMP-2 and MMP-9 activity was determined based on three independent experiments (b). The expression levels of secretory MMP-2 and MMP-9 in the culture medium (c) and in the cell lysates (d) were determined by Western blot assay.

Article Snippet: Human renal proximal tubular epithelial cells (hRPTECs) cell line (ATCC® CRL-4031TM) was cultured in DMEM/F-12 1:1 (GibcoTM 11330032) supplemented with 1% fetal bovine serum (GibcoTM 10437028) and the hTERT immortalized RPTEC growth kit (ATCC® ACS-4007TM).

Techniques: Expressing, Cell Culture, Solvent, Control, Activity Assay, Zymography, Western Blot

Journal: Matrix Biology Plus

Article Title: A proteome comparison between human fetal and mature renal extracellular matrix identifies EMILIN1 as a regulator of renal epithelial cell adhesion

doi: 10.1016/j.mbplus.2019.100011

Figure Lengend Snippet: Effect of EMILIN1 or Fibrillin1 depletion from the extracellular matrix on renal epithelial cell growth and migration. (A) ki67/DAPI ratio as measure for cell proliferation at 24, 48 or 72 h post seeding in HRPTECs cultured on siSHAM, siEMILIN1 and siFBN1 ECM. Shown is mean ± SEM. N ≥ 26 fluorescent images derived from N = 3 samples. (B) Viability measurements using the PrestoBlue assay after 24, 48 or 72 h post-seeding in HRPTECs on siSHAM, siEMILIN1 and siFBN1 ECM. Shown is mean ± SEM. N = 6 assays. (C) DNA abundance measurements using the PicoGreen assay after 24, 48 or 72 h post-seeding in HRPTECs on siSHAM, siEMILIN1 and siFBN1 ECM. Shown is mean ± SEM. N = 5 assays. Quantified live-cell migration assay results showing velocity and covered and Euclidean distance of HK2 cells on either siEMILIN1 (D-F) and siFBN1 (G-I) ECM compared to cells on siSHAM ECM. The migration of HK2 cells on the ECM layer was tracked overnight with a confocal microscope. Shown is mean ± SEM; *P < 0.05, ** P < 0.01. N = 60 individual cells tracked derived from N = 4 assays. Migration plots showing multiple tracks of individual HK2 cells on siSHAM (J), siEMILIN1 (K) and siFBN1 ECM (L).

Article Snippet: Primary Human Renal Proximal Tubular Epithelial Cells (HRPTECs) were purchased from ScienCell (#4100) and maintained in Epithelial Cell Medium (#4101; ScienCell).

Techniques: Migration, Cell Culture, Derivative Assay, Prestoblue Assay, Picogreen Assay, Cell Migration Assay, Microscopy

Journal: Matrix Biology Plus

Article Title: A proteome comparison between human fetal and mature renal extracellular matrix identifies EMILIN1 as a regulator of renal epithelial cell adhesion

doi: 10.1016/j.mbplus.2019.100011

Figure Lengend Snippet: Depletion of EMILIN1 from the ECM layer reduces paxillin area and stress fibers in renal epithelial cells. (A) Quantified results showing the paxillin area in HRPTECs on siSHAM or siEMILIN1 ECM during initial binding to the ECM (cultured for 2 h). Shown is mean ± SEM; ***P < 0.001. N = 30 fluorescent images derived from N = 3 samples. (B) Representative immunofluorescence images (400× magnification) of HRPTECs after 2 h of binding on siSHAM or siEMILIN1 ECM and stained for paxillin (green), F-actin (red) and DAPI (blue). Scale bar represents 20 μm (overview image, left) and 5 μm (zoomed-in image, right). Quantified results showing the ZO-1 (C) and F-actin area (D) in HK2 cells cultured to confluency on siSHAM or siEMILIN1 ECM. Shown is mean ± SEM; ***P < 0.001. N = 20 fluorescent Z-stacks derived from N = 4 samples. (E) Representative immunofluorescence Z-stacks (630× magnification) of HK2 cells cultured to confluency (cultured for 48 h) on siSHAM or siEMILIN1 ECM and stained for ZO-1 (green), F-actin (red) and DAPI (blue). Scale bar represents 20 μm (overview image, left) and 5 μm (zoomed-in image, right). (F) Colocalization of paxillin and F-actin in HK2 cells cultured to confluency on siSHAM or siEMILIN1 ECM. Shown is mean ± SEM; * P < 0.05. N ≥ 25 fluorescent Z-stacks, from N ≥ 5 samples. (G) Representative immunofluorescence Z-stacks (630× magnification) of HK2 cells cultured to confluency on siSHAM or siEMILIN1 ECM and stained for paxillin (green), F-actin (red) and DAPI (blue). Colocalization is displayed as yellow. Scale bar represents 15 μm (overview image, left) and 5 μm (zoomed-in image, right). (H) RhoA activation levels in HK2 cells cultured for 48 h on siSHAM or siEMILIN1 ECM. Shown is mean ± SEM; *P < 0.05. N = 5 assays.

Article Snippet: Primary Human Renal Proximal Tubular Epithelial Cells (HRPTECs) were purchased from ScienCell (#4100) and maintained in Epithelial Cell Medium (#4101; ScienCell).

Techniques: Binding Assay, Cell Culture, Derivative Assay, Immunofluorescence, Staining, Activation Assay