Journal: Frontiers in Cellular and Infection Microbiology

Article Title: An algal lectin griffithsin inhibits Hantaan virus infection in vitro and in vivo

doi: 10.3389/fcimb.2022.881083

Figure Lengend Snippet: GRFT inhibits rVSV-HTNV-GFP entry. The recombinant vesicular stomatitis virus harboring HTNV glycoproteins (rVSV-HTNV-G) and GFP were treated with varying concentrations of GRFT for 1 h, and then the mixture was used to infect Vero-E6 (A) or A549 (B) cells at an MOI of 1. At 24 h post-infection, cell nuclei were stained with Hoechst 33258 and observed under an inverted fluorescence microscope. Green, GFP, indicates the replication of rVSV-HTNV-G; blue, cell nuclei. (C,D). The dose–response curve shows the quantification of GFP-positive Vero-E6 (C) or A549 (D) cell area after GRFT treatment of rVSV-HTNV-G (normalized with the positive control, the first point in the figure shows the first GRFT treatment concentration instead of positive control). (E, F) Cell viability was measured 72 h after drug administration using the Cell Counting Kit-8 (CCK8), and absorbance for Vero E6 (E) cells and A549 (F) cells was measured at 450 nm using the BioTek HT collaborative instrument. A450 levels were subtracted from blank well background, and untreated cells were set at 100% viability. Data shown in the graphs are presented as the mean ± SD and are representative of three independent experiments performed in hexaplicate. GRFT, griffithsin; rVSV, recombinant vesicular stomatitis virus; HTNV, Hantaan virus; GFP, green fluorescent protein; MOI, multiplicity of infection.

Article Snippet: Hoechst 33258 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Recombinant, Virus, Infection, Staining, Fluorescence, Microscopy, Positive Control, Concentration Assay, Cell Counting

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: An algal lectin griffithsin inhibits Hantaan virus infection in vitro and in vivo

doi: 10.3389/fcimb.2022.881083

Figure Lengend Snippet: GRFT-pretreated cells reduce rVSV-HTNV-GFP and HTNV infection. (A, B) Vero-E6 cells (A) or A549 cells (B) were treated for 1 h with varying concentrations of GRFT and removed before being infected with rVSV-HTNV-G at an MOI of 1. At 24 h post-infection (hpi), the cell nuclei were stained with Hoechst 33258 and viewed under an inverted fluorescence microscope. Green, GFP, indicating the replication of rVSV-HTNV-G; blue, cell nuclei. (C, D) The quantitation of GFP-positive Vero-E6 (C) or A549 (D) cells after GRFT pretreatment of cells and infection with rVSV-HTNV-G (% normalized to the vehicle-only control) are shown in the dose–response curve. (E, F) The effects of GRFT antagonized HTNV infection in GRFT-pretreated cell group (Pre), or GRFT-treated virus group (Mix). Vero E6 (E) and A549 (F) cells were pretreated with 5 or 50 μg/ml of GRFT or vehicle and then infected with HTNV (MOI = 1), or equivalent HTNV was treated with 5 or 50 μg/ml of GRFT or vehicle for 1 h, and then the mixture was used to infect Vero E6 and A549 cells. At 72 hpi, the NP level within the cell was detected and normalized to GAPDH level. Data shown in the graphs are presented as the mean ± SDs and are representative of three independent experiments performed in triplicate. GRFT, griffithsin; rVSV, recombinant vesicular stomatitis virus; HTNV, Hantaan virus; MOI, multiplicity of infection; GFP, green fluorescent protein.

Article Snippet: Hoechst 33258 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Staining, Fluorescence, Microscopy, Quantitation Assay, Control, Virus, Recombinant

Journal: Free radical biology & medicine

Article Title: Drp1-regulated PARK2-dependent mitophagy protects against renal fibrosis in unilateral ureteral obstruction.

doi: 10.1016/j.freeradbiomed.2019.12.005

Figure Lengend Snippet: Fig. 7. Mdivi-1 inhibited hypoxia-induced PARK2-dependent mitophagy, increased mtROS production, and promoted TGFβ1 signaling in HK-2 cells. (A)Immunoblot analysis of Drp1 in mitochondria of HK-2 cells and (B) quantification of immunoblot analysis (n = 4 per group). (C)HK-2 cells were pretreated with vehicle or Mdivi- 1(5 μM) 1h before hypoxia exposure, and were cultured in hypoxic condition(1%O2) for 24 h. Immunoblot analysis of proteins in mitochondrial fraction of HK- 2 cells, and (D) densitometric analysis (n = 3 per group). (E)Representative images of MitoSOX/Hoechst staining of HK-2 cells and (F)quantification data of fluorescence density (n = 6 per group). Scale bar, 25 μm. (G) MnSOD activity of HK-2 cells analyzed by colorimetric activity assay(n = 6 per group).(H) MnSOD protein level detected by immunoblot analysis and(I) quantification data(n = 4 per group). (J) Immunoblot analysis and densitometry data of the protein level of (K)TGFβ1, (J)phosphorylation of smad2 and (L)smad3 in HK-2 cells (n = 4 per group). Error bars: SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: Living HK-2 cells were incubated in MitoSOX (5 μM) in HBSS and Hoechst (5 μg/ml,HY15631,MedChemExpress) for 30 min at 37 °C, and then observed under fluorescence microscope.

Techniques: Western Blot, Cell Culture, Staining, Activity Assay, Phospho-proteomics

Journal: Free radical biology & medicine

Article Title: Drp1-regulated PARK2-dependent mitophagy protects against renal fibrosis in unilateral ureteral obstruction.

doi: 10.1016/j.freeradbiomed.2019.12.005

Figure Lengend Snippet: Fig. 9. MitoTEMPO treatment attenuated hypoxia-induced mtROS production and TGFβ1 signaling in HK-2 cells. After transfected with siRNAs for 24 h, HK-2 cells were pretreated with vehicle or mitoTEMPO(100 μM) 4h before hypoxia exposure, and were then cultured in hypoxic conditions(1%O2) for 24 h. (A)Representative images of MitoSOX/Hoechst staining of HK-2 cells and (B)quantification data of fluorescence density (n = 3 per group). Scale bar, 25 μm. (C) Immunoblot analysis and densitometry data of phosphor the protein level of (D)TGFβ1, (E)phosphorylation of smad2 and (F)smad3 in HK-2 cells (n = 3 per group). Error bars: SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. NC, negative control; Norm, normoxia; Hypo, Hypoxia for 24 h; mT, mitoTEMPO.

Article Snippet: Living HK-2 cells were incubated in MitoSOX (5 μM) in HBSS and Hoechst (5 μg/ml,HY15631,MedChemExpress) for 30 min at 37 °C, and then observed under fluorescence microscope.

Techniques: Transfection, Cell Culture, Staining, Western Blot, Phospho-proteomics, Negative Control

Journal: Saudi Journal of Biological Sciences

Article Title: E74-like factor 3 suppresses microRNA-485-5p transcription to trigger growth and metastasis of ovarian cancer cells with the involvement of CLDN4/Wnt/β-catenin axis

doi: 10.1016/j.sjbs.2021.04.093

Figure Lengend Snippet: Upregulation of CLDN4 diminishes the suppressing role of ELF3 inhibition in OC progression. A, mRNA expression of CLDN4 after sh-ELF3 or LV-CLDN4 transfection was determined by RT-qPCR (one-way ANOVA,* p < 0.05 compared to sh-NC, # p < 0.05 compared to sh-ELF3 + LV-NC); B-C, viability (B) and proliferation (C) of OC cell lines determined by MTT and cell colony formation assays, respectively (one-way ANOVA, * p < 0.05); D-E, migration (D) and invasion (E) abilities of OC cells measured by scratch test and Transwell assay, respectively (one-way ANOVA, * p < 0.05); F, protein levels of EMT-related biomarkers (E-cadherin and vimentin) detected by western blot analysis (two-way ANOVA, * p < 0.05); G, protein levels of apoptosis-related factors (Bax and Bcl-2) in OC cells measured by ELISA kits; H, apoptosis of OC cells measured by Hoechst 33,258 staining (one-way ANOVA, * p < 0.05). Data were exhibited as mean ± SD from three independent experiments.

Article Snippet: In brief, 48 h after transfection, cell slides were prepared and fixed in 4% paraformaldehyde, and then stained with Hoechst 33,258 solution (HY-15623, MedChemExpress, Monmouth Junction, NJ, USA) avoiding light exposure for 30 min.

Techniques: Inhibition, Expressing, Transfection, Quantitative RT-PCR, Migration, Transwell Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal: Journal of Virology

Article Title: Peste des petits ruminants virus (PPRV) induces ferroptosis via LONP1-mediated mitochondrial GPX4 degradation in cell culture

doi: 10.1128/jvi.02310-24

Figure Lengend Snippet: PPRV induces ferroptosis in EECs. EECs were mock-infected or infected with PPRV (MOI = 5) for 24 or 48 h, Erastin (20 µM) as a positive control. ( A ) The percentages analysis of dead cells stained with trypan blue solution, scale bar = 100µm. ( B ) Relative levels of cell viability were assayed by using CCK8. ( C ) Flow cytometry analysis of intracellular ferrous iron levels via stained with the fluorescent probe FerroOrange; the mean FerroOrange fluorescence intensity of each cell was quantified. ( D ) Representative images of fluorescence staining for ferrous iron levels in intracellular; the nucleus was stained with Hoechst 33258. Scale bars = 50 µm. ( E ) Flow cytometry analysis of intracellular lipid peroxidation levels using the fluorescent probe Liperfluo. ( F ) Representative images of fluorescence staining for lipid peroxidation levels in intracellular; the nucleus was stained with Hoechst 33258. Scale bars = 50 µm. ( G and H ) Detection of MDA concentration in cell lysates. ( I ) Representative images of normal mitochondria or dysfunctional mitochondria are shown under the TEM. Scale bar = 500 nm; the diameter of the mitochondria was quantified. ( J ) The percentages analysis of dead cells stained with trypan blue solution, scale bar = 100 µm. The data are given as mean ± SD from three independent experiments. P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control. * P < 0.05; ** P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Ferrostatin-1 and Erastin were purchased from Topscience (Hoechst 33258 [Solarbio]).

Techniques: Infection, Positive Control, Analysis, Staining, Flow Cytometry, Fluorescence, Concentration Assay, Comparison, Control, FACS, Transmission Electron Microscopy

Journal: Journal of Virology

Article Title: Peste des petits ruminants virus (PPRV) induces ferroptosis via LONP1-mediated mitochondrial GPX4 degradation in cell culture

doi: 10.1128/jvi.02310-24

Figure Lengend Snippet: PPRV regulated the GPX4-Nrf2-Keap1 pathway by downregulating LONP1. ( A ) EECs were treated with Erastin (15, 20, or 25 µM) or infection with PPRV (MOI  = 5) for 48 h, Western blotting analysis of LONP1 and GPX4 and β-actin/Actin expression. Western blotting analysis of LONP1 expression in EECs transfected with siLONP1 or siNC for 48 h ( B ) and EECs were transfected with pcDNA3.1-LONP1 plasmids or pcDNA3.1 plasmids ( C ). ( D ) MitoSOX levels of control (Mock or NC), transfected with siLONP1 and PPRV infected with or without transfected with pcDNA3.1-LONP1 plasmid cells were detected by MitoSOX staining and analyzed by flow cytometry. Then, the mean MitoSOX fluorescence intensity of each cell was quantified. ( E ) Detection of MDA concentration in cell lysates. ( F ) Fluorescence analysis of the mitochondrial lipid peroxidation levels of control (NC), transfected with siLONP1, PPRV-infected cells, and PPRV infection treated with Fer-1 (20 µM) or transfected with pcDNA3.1-LONP1 plasmids and Erastin (20 µM) treatment cells stained for Liperfluo probe, Mito-Tracker, and Hoechst 33258. Scale bar = 10 µm. Colocalization analysis of immunofluorescence images using the colocalization plugin, which calculates Pearson’s correlation coefficient. ( G and H ) Western blotting and quantification of representative protein levels of LONP1, GPX4, Nrf2, Keap1, and β-actin/Actin (loading control) of EECs with knockdown and overexpression of LONP1 ( G ), control cells, PPRV-infected cells, and PPRV infection treated with Fer-1 (20 µM) or transfected with pcDNA3.1-LONP1 plasmid cells ( H ). ( I and J ) Western blotting analysis of GPX4 and VDAC1 proteins in mitochondrial fractions ( I ) and cytoplasmic fractions ( J ) from EECs PPRV infected and PPRV infection treated with Fer-1 (20 µM) or transfected with pcDNA3.1-LONP1 plasmid cells. ( K ) Western blotting analysis of GPX4 expression in EECs transfected with pcDNA3.1-GPX4 or pcDNA3.1 plasmids for 48 h. ( L ) Western blotting analysis of LONP1, GPX4, and β-actin/Actin protein levels in mock-infected and PPRV-infected cells with or without the transfection of pcDNA3.1-GPX4 or pcDNA3.1 plasmids at 48 h. The data are given as means ± SD from three independent experiments. P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Ferrostatin-1 and Erastin were purchased from Topscience (Hoechst 33258 [Solarbio]).

Techniques: Infection, Western Blot, Analysis, Expressing, Transfection, Control, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Concentration Assay, Immunofluorescence, Knockdown, Over Expression, Comparison

Journal: Journal of Virology

Article Title: Peste des petits ruminants virus (PPRV) induces ferroptosis via LONP1-mediated mitochondrial GPX4 degradation in cell culture

doi: 10.1128/jvi.02310-24

Figure Lengend Snippet: PPRV H, N, and F proteins are involved in ferroptosis induction. EECs were transfected with plasmids expressing various HA- or Flag-tagged viral proteins for 48 h and then subjected to western blotting with antibody against HA or Flag for the analysis of the expression of PPRV P, N, H, V, C, and F proteins in EECs ( A ). ( B ) Western blotting analysis of the protein levels of representative protein levels of ACSL4, LONP1, GPX4, and β-actin /Actin (loading control) of EECs with overexpression of PPRV H, N, V, C, P and F proteins, control cells, and PPRV-infected cells. ( C ) Western blotting analysis of LONP1 and GPX4 expression in EECs transfected with different doses of PPRV-N plasmids for 48 h. ( D ) The relative levels of cell viability were assayed by using cell CCK8. ( E ) Detection of MDA concentration in cell lysates. Representative images of EECs stained with FerroOrange ( F ) and with Liperfluo ( G ), treatment with Erastin and PPRV-infected cells as the positive control. Cell nucleus was counterstained with Hoechst 33258. Scale bars = 50 µm. The fluorescence intensity was quantified by ImageJ software. The data are given as means ± SD from three independent experiments. P values were calculated using Student’s t test. An asterisk indicates a comparison with the indicated control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Ferrostatin-1 and Erastin were purchased from Topscience (Hoechst 33258 [Solarbio]).

Techniques: Transfection, Expressing, Western Blot, Analysis, Control, Over Expression, Infection, Concentration Assay, Staining, Positive Control, Fluorescence, Software, Comparison, Immunofluorescence