Fig 2: Expression of ORF8 shows the downregulation and insufficient S1/S2 processing of the spike protein in a dose-dependent manner. (A) Spike protein immunostaining of Spike pseudovirus-packaging cells was performed. One representative result is shown, and the yellow color indicates the spike signal. EV: empty vector. (B) Immunoblots of Spike pseudovirus-packaging cells were obtained with antibodies specific to the spike protein and FLAG tag to visualize the protein level of the spike protein and ORF8. An antibody specific to Hsp90 served as a loading control. The scatter plot shows the relative intensity of the signals quantified by the ImageJ software from three independent experiments. The cleaved S1 and S2 fragments (between 100 to 130 kDa) were selected and determined as “S1 + S2 spike protein” and all fragments visualized above 100 kDa (containing the cleaved S1/S2 fragments and the full-length spike protein) were all selected and evaluated as “total spike protein” for quantification in this figure. In order to visualize the spike signal in ORF8-transfected samples together with EV-transfected samples in the same blot, the intensity of S1/S2 fragment of EV-transfected might reach plateau level then the real intensity might be underestimated. So the true efficiency of S1/S2 cleavage in EV-transfected cells might be much higher than the data shown here. (C) Immunoblots of cells transfected with ORF8 or ORF3a in combination with different spike variants. Here, an antibody specific to the FLAG tag indicates the protein levels of ORF8 and ORF3a. WT: Spike obtained from the original Wuhan strain; D614G: WT spike protein with the D614G mutation; D614G/N501Y: WT spike protein with the D614G and N501Y mutations. (D) Immunoblots of HEK293T cells transfected with different amounts of ORF8-expressing plasmids were obtained 3 days post-transfection. Here, an antibody against actin served as the loading control. Empty: empty vector does not encode ORF8. (E) FACs analyses of surface spike protein were performed using HEK293T cells cotransfected with spike-encoding plasmid together with pLAS2-based plasmid carrying ORF8 or empty vector at 1–9 ratio at 3 days post-transfection. The data are shown as the means ± SDs (error bars). Here, the paired Student’s t-test was used, and p < 0.05 indicates a statistically significant difference; *p < 0.05, **p < 0.01, ***p < 0.001. The scale bar in each figure represents 100 μm.

Fig 3: Analysis of factors involved in regulation of cell cycle in both Pax7ko and Pax7wt ESCs and MEFs. (A). RT-qPCR analysis of Ccnd1, Ccne1, Ccna2, Ccnb2, Cdkn1a, Cdkn1b, Cdkn1c, Plk1, Cdc34, and Mcm5 transcript levels in undifferentiated ESCs, EBs at day 2, 7 and EB outgrowths at day 21 of differentiation, as well as asynchronously dividing, quiescent and synchronously cycling MEFs. Data are shown as CT values, which were normalized against those of Actb mRNA level. Data are represented as the percentage of expression observed in mouse embryos at day 13.5 of development. For each genotype 2 ESC and 3 MEF lines were analyzed, graphs represent the mean values. P-values: *<0.05, ** <0.01, *** <0.001. (B). Western blotting analysis of cyclin E1 levels in ESCs, EBs at day 2 and 5, and EB outgrowths at day 14 of differentiation. Probing with anti-Hsp90 antibody was used as a loading control. Graph represents optical density of cyclin E bands compared to density of Hsp90 bands (optical density of Hsp90 was taken as 100 arbitrary units).

Fig 4: Selected gene expression in teratomas obtained from Pax7+/+ and Pax7-/- ESCs. a Expression of mRNAs encoding pluripotency marker (Nanog), marker of primitive streak—mesodermal cell marker (Brachyury), and markers of myogenic cells (Myod1, MyoG) (for each genotype n = 7). b Expression of mRNAs encoding embryonic myoblast markers (Pax3, Nfatc4) (for each genotype n = 7). c Expression of mRNAs encoding fetal myoblast markers (Nfix, Eno3, Mck, Mef2a, Itga7) (for each genotype n = 5 to n = 9). Expression was related to the levels observed in 13.5 d.p.c. mouse embryo (E13.5; n = 3), and normalized to mRNA encoding ß-actin (Actb). d–g Western blot analysis of Pax3, MyoD, myogenin, and Mck in teratomas obtained from three Pax7+/+ ESC lines and three Pax7-/- ESC lines (for each genotype n = 3). Hsp90 level was used as a loading control. Graph represents optical density of Pax3, MyoD, Myogenin, and Mck bands compared to density of corresponding Hsp90 bands (optical density of Hsp90 was accounted as 100%, OdU optical density, arbitrary units). White bar—values for Pax7+/+ teratomas, gray bar—values for Pax7-/- teratomas. Data characterized by non-normal distribution (c, Eno3) was normalized. Data are presented as mean ± SD. Stars represent results of Student’s unpaired two-tailed t test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig 5: Analysis of WNT secretion inhibition and WNT pathway activity in mouse ESCs cultured in medium supplemented either with FCS or SR. (A) Expression of Nanog, Pax6, Gata4, Tbxt, and Msgn1 in control and IWP-2-treated EBs. (B) Representative images of TUJ and TNNT immunodetection in outgrowths derived from control and IWP-2 treated EB5 cultured in the presence of FCS. Analyzed proteins are shown in red; DNA in blue. Scale bar: 50 µm. The graph shows the mean proportion of cells expressing indicated proteins in outgrowth. (C) Expression of Axin2 in undifferentiated ESCs and EBs. (D) Level of active (non-phosphorylated ß-CATENIN; ABC) and HSP90 in EBs. HSP90 was used as a loading control. Relative protein quantity ABC/HSP90 is shown on the graph. (E) Expression of Alcam1 in undifferentiated ESCs and EBs. (F) Level of phosphorylated JNK (pJNK) and TUBULIN in EBs. TUBULIN was used as a loading control. Relative protein quantity pJNK/TUBULIN is shown in the graph. (A,C,E) For qPCR analyses ß-actin was used as a reference gene. RQ = 1 for the expression level detected in a 13.5-day-old mouse embryo. The average ?Cq for reference sample is shown on each graph. Data presented as means of three independent experiments with standard deviations; * p < 0.05; ** p < 0.01; *** p < 0.001.