HCMV infection downregulates GPX4 and stimulates lipid peroxidation but does not induce ferroptosis Figure 6

HCMV-induced lipid peroxidation is sensitive to ferrostatin-1 but not GPX4. (A) Western blot analysis of lysates from MRC-5 cells expressing an empty vector (Vec) or GPX4 and either uninfected or infected with a GFP-tagged TB40/E-WT virus (MOI = 3) and harvested at 96 hpi. Blots were probed for GPX4, IE1, IE2, and actin as a loading control. The values under the GPX4 blot denote quantification of each GPX4 band in the blot, relative to the GPX4 band in the uninfected vector cells. (B) Infectious titers at 120 hpi of MRC-5 cells expressing an empty vector or GPX4 and infected with TB40/E-WT at MOI = 3. Results are from two independent experiments, n = 4. (C) Analysis of HCMV spread in MRC-5 cells expressing an empty vector or GPX4 and infected at a low MOI (0.05) with a GFP-tagged TB40/E-WT virus (TB40/E-IE2AGFP). The fluorescence area (μm2) was measured every 3 days post-infection to monitor spread. Results are from two independent experiments, n = 5. (D) Lipid peroxidation measured by C11 BODIPY FITC fluorescence shift in MRC-5 cells expressing either an empty vector or GPX4. Lipid peroxidation was measured in both uninfected cells and cells infected as described in panel B at 96 hpi. Results are from two independent experiments, n = 6. (E) Infectious titers at 120 hpi of MRC-5 cells infected as described in panel B and treated with either DMSO or 1 μM ferrostatin-1 (Fer1) at both 3 and 72 hpi. Results are from two independent experiments, n = 3. (F) Lipid peroxidation measured at 72hpi by C11 BODIPY FITC fluorescence shift in MRC-5 cells either uninfected or infected as described in panel B that were treated for 48 hours with either DMSO or 1 μM ferrostatin-1 (Fer-1). As a positive control, uninfected MRC-5 cells were treated with 1 μM RSL3 with either DMSO or 1 μM ferrostatin-1 (Fer-1) for 4 hours before collection. Results are from two independent experiments, n = 6. For the above panels, * indicates P < 0.05 and ns = not significant.

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Norbury, Christopher (2024). HCMV infection downregulates GPX4 and stimulates lipid peroxidation but does not induce ferroptosis Figure 6 [Data set]. Scholarsphere.

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Work Title HCMV infection downregulates GPX4 and stimulates lipid peroxidation but does not induce ferroptosis Figure 6
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  1. Christopher Norbury
License CC BY 4.0 (Attribution)
Work Type Dataset
Publication Date December 20, 2024
Deposited December 20, 2024

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    Description
    • HCMV-induced lipid peroxidation is sensitive to ferrostatin-1 but not GPX4. (A) Western blot analysis of lysates from MRC-5 cells expressing an empty vector (Vec) or GPX4 and either uninfected or infected with a GFP-tagged TB40/E-WT virus (MOI = 3) and harvested at 96 hpi. Blots were probed for GPX4, IE1, IE2, and actin as a loading control. The values under the GPX4 blot denote quantification of each GPX4 band in the blot, relative to the GPX4 band in the uninfected vector cells. (B) Infectious titers at 120 hpi of MRC-5 cells expressing an empty vector or GPX4 and infected with TB40/E-WT at MOI = 3. Results are from two independent experiments, n = 4. (C) Analysis of HCMV spread in MRC-5 cells expressing an empty vector or GPX4 and infected at a low MOI (0.05) with a GFP-tagged TB40/E-WT virus (TB40/E-IE2AGFP). The fluorescence area (μm2) was measured every 3 days post-infection to monitor spread. Results are from two independent experiments, n = 5. (D) Lipid peroxidation measured by C11 BODIPY FITC fluorescence shift in MRC-5 cells expressing either an empty vector or GPX4. Lipid peroxidation was measured in both uninfected cells and cells infected as described in panel B at 96 hpi. Results are from two independent experiments, n = 6. (E) Infectious titers at 120 hpi of MRC-5 cells infected as described in panel B and treated with either DMSO or 1 μM ferrostatin-1 (Fer1) at both 3 and 72 hpi. Results are from two independent experiments, n = 3. (F) Lipid peroxidation measured at 72hpi by C11 BODIPY FITC fluorescence shift in MRC-5 cells either uninfected or infected as described in panel B that were treated for 48 hours with either DMSO or 1 μM ferrostatin-1 (Fer-1). As a positive control, uninfected MRC-5 cells were treated with 1 μM RSL3 with either DMSO or 1 μM ferrostatin-1 (Fer-1) for 4 hours before collection. Results are from two independent experiments, n = 6. For the above panels, * indicates P < 0.05 and ns = not significant.
    Publication Date
    • 2024-12-20
  • Added Creator Christopher Norbury
  • Added Maddie Martin 2024-02-02 09h18m54s.scn
  • Added 020124 GPX4 OE protein calcs.xlsx
  • Added 8-22-22 GPX4 OE titers .xlsx
  • Added 11-15-22 GPX4 OE Low MOI titers.xlsx
  • Added 8-10-22 GPX4 OE DMSO, iFSP1 titers.xlsx
  • Added 091723 GPX4 OE C11 BODIPY combined.xlsx
  • Added GPX4 OE iFSP1 spread assays combined.xlsx
  • Added 012424 GPX4 OE C11 BODIPY combined.xlsx
  • Added 031723 RSL3, Erastin, iFSP1, Ferrostatin1 titer plates.xlsx
  • Added 041423 RSL3 Erastin and Ferrostatin titers.xlsx
  • Added 020724 C11 BODIPY Fer DFO combined.xlsx
  • Added Figure 6.pdf
  • Updated License Show Changes
    License
    • https://creativecommons.org/licenses/by/4.0/
  • Added Fig6 readme.txt
  • Published
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