Fig 2: Monocyte infiltration and extensive microglia activation in COVID-19 brains. a Histology shows neuronophagia, microglial nodule and perivascular inflammation in COVID-19 brain tissues. b Immunohistochemical staining for IBA1 shows reactive activation of microglia. c Immunohistochemistry shows CD16+ and CD14+ inflammatory cells infiltrating perivascular space and parenchyma. d Quantitative analysis of the type and distribution of immune cells in the brains of all COVID-19 patients. In addition to CD16+ and CD14+ infiltrating cells, lymphocytic infiltration around the vessel is also seen (please see Supplementary Fig. S1). e The mononuclear cells (black arrows) in the microvessel (*) walls and perivascular spaces are positive for monocyte biomarkers HLA-DR, CCR7, CD141 and CD11c. f Scheme of proteomics study. g Principal component analysis of 15 samples based on quantitative profiles of proteins of brain tissues. Red and blue dots represent brain samples from COVID-19 patients and healthy controls. The red color represents control brains, while the blue color represents COVID-19 brains. h Volcano plots of the −log10 p-value vs. the log2 protein abundance comparisons between brains from normal subjects and those with COVID-19. Proteins outside the significance threshold lines (−log10 (p-value) > 2 and log2 (fold change) > 1 or <−1) are in red (upregulated) or blue (downregulated). The p values are calculated for proteins identified in brain tissues from the COVID-19 patients and healthy controls. i Proteomic analysis showing the activated monocyte-related pathway changes and release of inflammatory factors

Fig 3: EBOV VP35 promotes the transcription of CREB1-directed coagulation-related genes.a HepG2 cells cotransfected with pGL-CRE-Luc, pRL-TK, and the indicated amounts of Flag-VP35 were treated with or without 25 μM FSK for 4 h. The luciferase activity of the cell lysates was analyzed. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The mean ± s.e.m. from three independent assays is presented (n = 3; ***P < 0.001). b, c HUVECs (b) and WT or AKIP1−/− HepG2 cells (c) infected with Ad-VP35 (MOI = 10) (b) or live EBOV (MOI = 1) (c) for 48 h were treated with or without 10 μM H89 or 1 μM 666-15 for another 24 h. THBD and SERPINB2 mRNA levels were determined by qRT-PCR. Differences between the two groups were evaluated using the two-sided unpaired Student’s t-test. Data were presented as mean ± s.e.m. (n = 3; ***P < 0.001). d WT or Akip1−/− mice were intravenously injected with Ad-VP35 or Ad-null (2 × 109 PFU) twice at an interval of 24 h. Six days post the first infection, the liver tissues were analyzed by immunohistochemistry staining with an anti-Thrombomodulin (TM) antibody. e, f Mice were infected with Ad-VP35 or Ad-null (3 × 109 PFU), treated with 666-15 (2 mg/kg) or solvent and then challenged with or without LPS. The tail bleeding time was determined (n = 8) (e), and a mouse survival curve is shown in (f) (n = 9). Differences between the two groups were evaluated using the two-sided unpaired Student’s t-test (e). Survival curves were analyzed by log-rank test (f). All data from two independent experiments are presented as the means ± s.e.m. (ns not significant; *P < 0.05; **P < 0.01).

Fig 4: Reduced total and phosphorylated IGF1R protein levels in differentiated DCs. (A) Human leukemic THP-1 cells were differentiated to immature DCs by treatment with 100 ng/ml IL-4 and 100 ng/ml GM-CSF for 5 days and then differentiated to DCs by treatment with 200 ng/ml IL-4, 100 ng/ml GM-CSF, 20 ng/ml TNFα, and 200 ng/ml Ionomycin for 2 days. THP1 scale bar = 50 μm, immature DCs scale bar = 500 μm, DCs scale bar = 500 μm. (B) DC marker expression via CD11b and CD141 in THP-1, ImDC, and mDC. (C) THP-1 and THP-1 cells were treated with 200 ng/ml IL-4, 100 ng/ml GM-CSF, 20 ng/ml TNFα, and 200 ng/ml Ionomycin for 72 hours and were incubated with IGF1 for 10 minutes before harvest, after which cell extracts were prepared. Proteins were separated through SDS-PAGE, followed by electrophoretic transfer and incubation with antibodies against tIGF1R, pIGF1R, tAKT, and pAKT. (D) Scanning densitometry analysis of tIGF1R (blue bars) and pIGF1R (red bars) levels in THP-1 and DCs. A value of 100% was given to the expression level of untreated THP-1 cells. Levels of tubulin were measured as a loading control. *P < .05. Bars represent SEM values. (E) Scanning densitometry analysis of pAKT levels in THP-1 and DCs. A value of 100% was given to the expression level of untreated THP-1 cells. Levels of tubulin were measured as a loading control.*P < .05. Bars represent SEM values. The graphs represent average of three independent experiments. (F) Expression of tIGF1R and pIGF1R with (green) or without (blue) IGF1 treatment in THP-1 cells.

Fig 5: Positive immunohistochemical staining. (A-F) IGFIR, p53, Ki67, BRCA1, CD141, and CD1c in early stage of serous subtypes ovarian carcinoma (×40), respectively. (G-L) IGFIR, p53, Ki67, BRCA1, CD141, and CD1c in advanced stage of serous subtypes ovarian carcinoma (×40), respectively. Scale bar = 50 μm.