Fig 2: Protein expression analysis of cell cultures under normoxic and hypoxic conditions. (A) Western blot analysis of IGFBP3, HIF-1a and HIF-2a, HO-1, VHL, and GAPDH expressions in P0, P4, P4 transfectants, and A549 under normoxic and hypoxic conditions. HSP90 was added as the control. (B) Western blot results were transformed into bar figures using Image Studio Lite version 5.2. Signal strengths were quantified and normalized with HSP90. P0 had a higher IGFBP3 expression than P4. In P0 and P4-I, IGFBP3 expression was increased under hypoxia. Accumulation of HIF-1a or HIF-2a correlated with IGFBP3 expressions: a higher IGFBP3 was with a stronger HIF-1a. Both HO-1 and VHL expressions were increased under hypoxia. Each sample was assayed in triplicates, and the experiment was repeated three times independently. (The error bars represent the SDs; **P < 0.001, ***P < 0.0005). Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Fig 3: TBM remarkably ameliorated DOX-evoked oxidative stress in H9c2 cells. (a, b) Flow cytometry was used to estimate the ROS levels in H9c2 cells (n = 4). (c–e) The level of MDA and the activity of SOD and GSH-PX in H9c2 cells were detected by commercial kits (n = 4). (f–h) The mRNA levels of Nrf2, Ho-1, and Nqo1 in H9c2 cells determined by qPCR (n = 5). (i–m) Representative western blotting results for Nrf2, HO-1, and NQO1 in vitro (n = 3). Values indicate the mean ± SD. *P < 0.05 versus the CON group and the TBM group; #P < 0.05 versus the DOX group; ns (not significant) versus the CON group.

Fig 4: (A) Immunostaining for HO-1: note the positivity of a few inflammatory cells available mainly subepithelial in the skin of a patient positive for SARS-CoV-2 (Immunohistochemistry, Original Magnification: 40×). The light brown color is linked to “background noise” of the immunohistochemical reaction. (B) Photomicroghaph of skin biopsy of a patient negative for SARS-CoV-2 with higher expression of HO-1 in the epidermis and of inflammatory cells in subepithelial disposition (Immunohistochemistry, Original Magnification: 100×).

Fig 5: Immunohistochemistry staining of xenografts on 12, 15, and 28 d after the tumor implantation. (A) Heterotransplantation tumors P4-pBIG2i and P4-pBIG2i-hIGFBP3 at 12, 15, and 28 d post-tumor implantation on IGFBP3 (100 ×), HIF-1a (40 × and 100 ×), HIF-2a (40 × and 100 ×), HO-1 (100 ×), and VHL (100 ×). P4-pBIG2i xenograft showed no IGFBP3 expressions, while P4-pBIG2i-hIGFBP3 showed a strong IGFBP3 expression after adding doxycycline (started on d 8). HIF-2a increased over time in P4-pBIG2i xenograft, but it only accumulated after 28 d in P4-pBIG2i-hIGFBP3 xenograft. Xenograft tumors without IGFBP3 expressed HIF-2a majorly, especially under a prolonged tumor growth in hypoxic environments. (B) Quantitative analysis of immunohistochemical staining. Images of IHC were taken by Motic Image Plus 3.0 software. The signals of IHC were quantified and analyzed by ImageJ 1.53 k software. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.