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. 2021 Aug 25;11:706252. doi: 10.3389/fcimb.2021.706252

Figure 5.

Figure 5

Disrupting the M-N interaction abolishes the enhancing activity of N on M-induced apoptosis. (A) The amino acid sequences of TAT, NP1-6, and MP3. (B) HEK293T cells were transfected with FLAG-SARS-CoV-2 M and HA-SARS-CoV-2 N and treated with TAT, NP1-NP6, and TM3, respectively. After 24 h, cells were collected for co-immunoprecipitation (with anti-FLAG) and Western blotting (with anti-FLAG or anti-HA). (C) HEK293T cells were transfected with FLAG-SARS-CoV-2 M and FLAG-SARS-CoV-2 M plus HA-SARS-CoV-2 N, respectively. Cells expressing FLAG-SARS-CoV-2 M plus HA-SARS-CoV-2 N were treated with NP3 and MP3, respectively. After 24 h, cells were collected for Western blotting with antibodies to the indicated proteins. (D) HEK293T cells were transfected with FLAG-SARS-CoV-2 M, GFP-PDK1 together with HA-SARS-CoV-2 N and treated with TAT, NP3, and TM3, respectively. After 24 h, cells were collected for co-immunoprecipitation (with anti-GFP or IgG) and Western blotting (with anti-FLAG, anti-HA, or anti-GFP). (E) HEK293T cells were transfected with GFP-PDK1, HA-Akt, Myc-SARS-CoV-2 M together with FLAG-SARS-CoV-2 N and treated with TAT, NP3, and TM3, respectively. After 24 h, cells were collected for co-immunoprecipitation (with anti-GFP, or IgG) and Western blotting (with anti-FLAG, anti-HA, anti-Myc, or anti-GFP). (F) Vero E6 and HepG2 cells were transfected with FLAG-SARS-CoV-2 M and SARS-CoV-2 M plus HA-SARS-CoV-2 N and treated with TAT, NP3, and TM3, respectively. After 24 h, cells were collected for Western blotting (with anti-FLAG, anti-HA, or anti-β-Actin) or stained with Annexin V-FITC/PI for flow cytometry analysis, and the percentage of apoptotic cells were measured. (G) HEK293T cells were transfected with FLAG-SARS-CoV-2 M and FLAG-SARS-CoV-2 M plus HA-SARS-CoV-2 N, respectively. Cells expressing FLAG-SARS-CoV-2 M plus HA-SARS-CoV-2 N were treated with NP3 and MP3, respectively. After 24 h, cells were collected for Western blotting with antibodies to the indicated proteins. ***P < 0.001 by two-tailed Student’s t-test. ns, non significant. The densities of blots were analyzed with ImageJ software.