HCMV infection downregulates GPX4 and stimulates lipid peroxidation but does not induce ferroptosis Figure 5

FSP1 knockdown does not impair infection or induce ferroptosis. (A) Western blot analysis of lysates from uninfected MRC-5 cells expressing control shRNA (green fluorescent protein [GFP]) or shFSP1 (shRNA against FSP1). Blots were probed for FSP1 and p115 as a loading control. The values under the blot denote quantification of each FSP1 band in the blot, relative to the FSP1 band in shGFP cells. (B) Infectious titers at 120 hpi from MRC-5 cells expressing GFP control shRNA or shFSP1 and infected with TB40/E-WT at MOI = 3. Results are from seven independent experiments, n = 18. (C) Lipid peroxidation was measured by C11 BODIPY FITC fluorescence shift in MRC-5 cells expressing shGFP or shFSP1 and either uninfected or infected as described in panel B at 96 hpi. MRC-5 cells treated with RSL3 (1 μM) were used as a positive control. Data are shown relative to uninfected shGFP cells. Results are from two independent experiments, n = 6. (D) ATP levels in MRC-5 cells infected as described in panel B at 96 hpi with cells expressing scrambled shRNA or shFSP1. Data are shown relative to shSCR cells. Results are from two independent experiments, n = 9. (E) LDH release in uninfected MRC-5 cells expressing shRNA against GFP or FSP. LDH release is shown as the percent of maximum LDH in uninfected control cells. Results are from three independent experiments, n = 9. (F) LDH release in MRC-5 cells expressing shRNA against GFP or GPX4 and infected with HCMV (as in panel A) and measured at 96 hpi. LDH release is shown as the percent of maximum LDH in infected cells. For the above panels, * indicates P < 0.05 and ns = not significant. Results are from three independent experiments, n = 9. 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 5 [Data set]. Scholarsphere.

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Work Title HCMV infection downregulates GPX4 and stimulates lipid peroxidation but does not induce ferroptosis Figure 5
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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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  • Updated Description, Publication Date Show Changes
    Description
    • FSP1 knockdown does not impair infection or induce ferroptosis. (A) Western blot analysis of lysates from uninfected MRC-5 cells expressing control shRNA (green fluorescent protein [GFP]) or shFSP1 (shRNA against FSP1). Blots were probed for FSP1 and p115 as a loading control. The values under the blot denote quantification of each FSP1 band in the blot, relative to the FSP1 band in shGFP cells. (B) Infectious titers at 120 hpi from MRC-5 cells expressing GFP control shRNA or shFSP1 and infected with TB40/E-WT at MOI = 3. Results are from seven independent experiments, n = 18. (C) Lipid peroxidation was measured by C11 BODIPY FITC fluorescence shift in MRC-5 cells expressing shGFP or shFSP1 and either uninfected or infected as described in panel B at 96 hpi. MRC-5 cells treated with RSL3 (1 μM) were used as a positive control. Data are shown relative to uninfected shGFP cells. Results are from two independent experiments, n = 6. (D) ATP levels in MRC-5 cells infected as described in panel B at 96 hpi with cells expressing scrambled shRNA or shFSP1. Data are shown relative to shSCR cells. Results are from two independent experiments, n = 9. (E) LDH release in uninfected MRC-5 cells expressing shRNA against GFP or FSP. LDH release is shown as the percent of maximum LDH in uninfected control cells. Results are from three independent experiments, n = 9. (F) LDH release in MRC-5 cells expressing shRNA against GFP or GPX4 and infected with HCMV (as in panel A) and measured at 96 hpi. LDH release is shown as the percent of maximum LDH in infected cells. For the above panels, * indicates P < 0.05 and ns = not significant. Results are from three independent experiments, n = 9. For the above panels, * indicates P < 0.05 and ns = not significant.
    Publication Date
    • 2024-12-20
  • Added Creator Christopher Norbury
  • Updated
  • Added Maddie Martin 2023-08-31 13h53m19s.scn
  • Added Maddie Martin 2023-09-01 15h24m19s.scn
  • Added Maddie Martin 2023-06-29 11h06m52s.scn
  • Added Maddie Martin 2023-06-30 09h49m32s.scn
  • Added protein calcs FSP1 GPX4 KD.xlsx
  • Added ladder 2023-01-25 13h24m26s.scn
  • Added ladder 2023-01-26 15h24m04s.scn
  • Added 090123 FSP1 & GPX4 KD titers.xlsx
  • Added 100723 FSP1 KD titers and TB40WT JS.xlsx
  • Added 063023 GPX4 FSP1 KD titers .xlsx
  • Added 070523 FSP1 KD LDH Assay.xlsx
  • Added 052024 FSP1 KD LDH Assay.xlsx
  • Added 051623 LDH Assay FSP1 KD.xlsx
  • Added 082423 FSP1 KD LDH.xlsx
  • Added 092623 FSP1 KD LDH.xlsx
  • Updated
  • Added Figure 5.pdf
  • Added Fig5 readme.txt
  • Updated License Show Changes
    License
    • https://creativecommons.org/licenses/by/4.0/
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