Fig 2: Trp depletion upregulates WARS expression.(A) WARS mRNA expression measured by qRT-PCR in A172 glioblastoma cells, which exhibit constitutive TDO2 activity, cultured for 120 h in normal DMEM or with additional supplementation of 78 µM Trp after 72 h. (B) WARS2 mRNA expression measured by qRT-PCR in A172 glioblastoma cells under the conditions described in (A). (C) WARS mRNA expression measured by qRT-PCR in glioblastoma (A172, LN18, LN229) and ovarian carcinoma cells (SKOV-3) after 24 h of cultivation in medium containing 78 µM versus no Trp. (D) WARS protein levels in LN229 glioblastoma cells detected by Western blot after 48 h or 72 h of cultivation in medium containing 78 µM or no Trp. GAPDH served as loading control (E) WARS2 mRNA expression measured by qRT-PCR was not altered by the conditions described in (C). (F) WARS protein levels in the cytoplasmic or nuclear fraction of LN229 glioblastoma cells detected by Western blot after 48 h of cultivation in medium containing 78 µM or no Trp. GAPDH served as loading control for the cytoplasmic fraction, lamin A/C as loading control for the nuclear fraction. (G) WARS protein concentrations measured by ELISA in the supernatants of LN229 and A172 glioblastoma cells cultured for 48 h in the presence or absence of Trp. All data are expressed as mean ± s.e.m. Statistical significance is assumed at P < 0.05 (*P < 0.05, **P < 0.01, ***P <0.001).

Fig 3: NSP6 and ORF7a induced NF-?B activation. (A) HEK293T cells were transfected with each SARS-CoV-2 protein expression plasmid, along with pGL4-NF-?B-LucP2 and pNull-RLuc. The level of luciferase activity was determined at 24 h posttransfection. The firefly luciferase activity was normalized to Renilla luciferase activity. An empty plasmid was used as a control, and the value was set as 1. Treatment with TNF-a (5 ng/mL) was performed for 24 h as a positive control. Results are shown as the mean ± standard deviation (SD) of three independent experiments. Statistics were generated by comparison with empty plasmid. **, P < 0.01 (Student’s t test). (B) HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, the mRNA and protein levels were determined by RT-qPCR and Western blotting, respectively. The indicated mRNA levels were normalized to GAPDH expression. An empty plasmid was used as a control and set to 1. Results are shown as the mean ± SD of three independent experiments. Statistics were generated by comparison with empty plasmid. *, P < 0.05; **, P < 0.01 (Student’s t test). (C) For the in vitro kinase assay, HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, cells were lysed with 1× IP lysis buffer, followed by IP with the anti-HA antibody. The immunocomplex was mixed with GST-I?Ba and ATP-?-S in the kinase buffer. Phosphorylation of GST-I?B was detected by anti-thiophosphate ester antibody.

Fig 4: Effect of a3 or a5 integrin silencing on IL-6 and IL-8 secretion by A549 cells during interaction with H. capsulatum. A549 cells were transfected with a3 or a5 integrin-directed siRNA or with Negative Control (NC) siRNA for 24 h, and then, incubated with H. capsulatum yeasts for 16 h. After incubation with fungi, culture supernatants were collected for determination of IL-6 and IL-8 levels, and A549 cells were harvested, lysed, and analyzed by Western blot. (A) Silencing of a3 and a5 integrins was confirmed by Western blot. GAPDH was used as protein loading control. Blots are representative of three independent experiments. (B) IL-6 and IL-8 levels in culture supernatants were determined by ELISA. Values represent the mean of triplicate experiments ± the standard deviation. *p < 0.01 when compared to NC siRNA. Similar results were obtained from three independent experiments.

Fig 5: hTERC encodes an ORF. (A) Western blot analysis of HEK293T and VA13 cells for the presence of hTERP. GAPDH staining was used as a loading control. (B and C) Immunolocalization of hTERP in VA13 (B) and HEK293T (C); blue, DAPI-stained nuclei; red, hTERP; scale bars, 10 µm. (D) Levels of hTERC in VA13, VA13 vector, VA13 hTERCstU, VA13 hTERCstop, VA13 hTERCwt (VA13 expressed empty vector or wild-type or mutant forms of hTERC) and HEK293T. Mean ± SD was calculated for three biological replicates. (E–G) Immunofluorescence analysis of VA13 hTERCstU (E), VA13 hTERCstop (F) and VA13 hTERCwt (G) (VA13 cells expressed wild-type or mutant forms of hTERC); blue, DAPI-stained nuclei; red hTERP; scale bars, 10 µm. (H) Western blot analysis of VA13, VA13 vector, VA13 hTERCstU, VA13 hTERCstop and VA13 hTERCwt cells for the presence of hTERP. GAPDH staining was used as a loading control.