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. 2022 Jan 10;13:105. doi: 10.1038/s41467-021-27670-1

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a TMEM120A did not inhibit ZIKV entry in Huh7 cells. Huh7 cells stably expressing luciferase or TMEM120A were incubated with ZIKV at an MOI of 1 at 37 °C for 4 h and then harvested for RT-qPCR. b, c TMEM120A co-localized with ER marker calnexin. HEK293T cells were transiently transfected with plasmids expressing HA-FLAG-tagged TMEM120A. At 48 h posttransfection, cells were fixed and permeabilized for immunostaining using HA (red) antibody and calnexin (green) antibody (b). Scale bar, 10 μm. TMEM120A-Calnexin co-localization was quantified using Pearson’s correlation coefficient method. Cells expressing both Calnexin and TMEM120A were selected randomly for co-localization analysis by ImageJ software (c). d TMEM120A inhibits ZIKV replication. HEK293T cells were transfected with TMEM120A or luciferase for 24 h, then transfected with ZIKV RNA replicon. A total of 48 h post replicon transfection, cells were harvested for Renilla luciferase assay. e, f TMEM120A co-localized with STING. HEK293T cells were transiently transfected with plasmids expressing STING and HA-FLAG tagged luciferase or TMEM120A. 48 h posttransfection, cells were fixed and permeabilized for immunostaining using STING (red) antibody and FLAG (green) antibody (e). Scale bar, 30 μm. STING-TMEM120A or STING-luciferase co-localization was quantified using Pearson’s correlation coefficient method. Cells expressing both STING and TMEM120A or luciferase were selected randomly for co-localization analysis by ImageJ software (f). g TMEM120A interacts with endogenous STING in U87MG cells. U87MG cells stably expressing HA-FLAG tagged luciferase or TMEM120A were infected with ZIKV for 48 h and then harvested for FLAG-tag based immunoprecipitation and immunoblotting to detect the interaction between TMEM120A and endogenous STING using STING antibody. 10% of the input was run and blotted. Input, STING and vinculin blots and FLAG-luciferase/TMEM120A blots are from two gels with the same samples. h STING interacted with TMEM120A in HEK293T cells. HEK293T cells were transiently transfected with plasmids expressing N-terminal HA-FLAG tagged luciferase or STING and C-terminal V5-TMEM120A for 48 h. Cells were then collected for FLAG-tag-based immunoprecipitation and immunoblotting to detect the interaction between TMEM120A and STING using TMEM120A antibodies. 10% of the input was run and blotted. In input, TMEM120A and vinculin binds are from one blot and FLAG-luciferase or STING band is from another blot with the same samples of the same amount of loadings. Cellular ZIKV RNA level was normalized to the internal control GAPDH. RT-qPCR in (a) and Renilla luciferase data in (d) represent the mean ± SEM (n = 3 independent experiments). Quantification of immunostaining data in (c, f) represent the mean ± SEM (n = 20 cells per group). *P < 0.05, ***P < 0.001 (unpaired, two-sided Student’s t-test). ns: not significant. Exact P values and statistical parameters are provided in Source Data File.