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. 2024 Sep 7;81(1):386. doi: 10.1007/s00018-024-05370-5

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Effect of different SARS-CoV-2 variants on HSF1 activation and HSP70 expression in the host cell. A Immunoblot analysis of SARS-CoV-2 viral spike, pHSF1-Ser326, HSF1 and α-tubulin protein levels in Vero-hACE2-TMPRSS2 cells mock-infected or infected for 48 h with SARS-CoV-2 Wuhan strain (0.1 PFU/cell), or in Vero E6 cells exposed to heat-stress (HS, 43 °C, 40 min). B Schematic representation of the experimental design of SARS-CoV-2 variants infection of Vero-hACE2-TMPRSS2 or MRC5-hACE2 cells. Immunoblot analysis of human angiotensin-converting enzyme 2 (hACE2) protein levels in MRC-5 wild type or expressing the human ACE2-receptor (M-hACE2) is shown. C Vero-hACE2-TMPRSS2 cells were mock-infected or infected with Wuhan, Alpha, Delta (0.1 PFU/cell) or Omicron (0.5 PFU/cell) SARS-CoV-2 variants for 48 h. Equal amounts of whole-cell extracts (10 μl) were analyzed for levels of spike, HSF1-Ser326, HSF1 and β-actin by IB (top panels). The pHSF1/HSF1 ratio was determined after normalizing to β-actin and expressed as fold induction of the mock-infected control, which was arbitrarily set to 1 (bottom panel). D MRC5-hACE2 cells were mock-infected or infected with the SARS-CoV-2 Omicron BA.1 variant (0.1 PFU/cell) for 24 h. Equal amounts of whole-cell extracts (15 μl) were analyzed for levels of spike, HSF1-Ser326, HSF1 and β-actin (left panels). The pHSF1/HSF1 ratio was determined after normalizing to β-actin and expressed as fold induction of the mock-infected control, which was arbitrarily set to 1 (right panel). E, F Vero-hACE2-TMPRSS2 cells were mock-infected or infected with different SARS-CoV-2 variants as in C. Equal amounts of whole-cell extracts (20 μl) were analyzed for levels of spike, GRP94, HSP90, HSP70, HSP60, pHSF1-Ser326, HSF1 and α-tubulin by IB (E). Uncleaved S proteins (S0) and S1 subunits are indicated by arrows. Relative amounts of GRP94, HSP90, HSP70, HSP60 and pHSF1 were determined after normalizing to α-tubulin and expressed as fold induction of the mock-infected control, which was arbitrarily set to 1 (F). C, D, F n = 4 (two technical replicates over two biological replicates)