Intercellular trafficking of fusion proteins VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs.

<p>COS-1 cells transfected with 0.5 µg pVP22-EGFP (A–D and I–L) and pEGFP-VP22 (E–H and M–P) DNA were trypsinized 24 hours after transfection and were co-plated with MSCs (A–H) or BrdU labeled MSCs (I–P) at a ratio of 1∶20. The co-cultured cell population were incubated for another 24 hours and were stained with anti-T-antigen (C, G, K and O) and anti-BrdU (I and M) antibodies. EGFP signals were detected not only in T-antigen positive COS-1 cells but also in the T-antigen negative and/or BrdU positive MSCs (arrows in D, H, L and P), demonstrating the spread of VP22-EGFP and EGFP-VP22 fusion proteins. Scale bar represents 20 µm.</p

The expression of VP22 in COS-1 cells.

<p>COS-1 cells were transfected with 0.5 µg pEGFP-N1 as mock or pVP22-myc and immunostained with anti-myc antibody 48 h after transfection. The anti-myc antibody showed no cross-reaction with mock transfected cells (A–C). The myc-tagged VP22 could be expressed in COS-1 cells (D–F). An intercellular transportation of VP22 protein could be inferred from the specific VP22 protein intercellular distribution: higher cytoplasmic expression in the center cells (arrows) and lower nucleic expression in the surrounding cells (arrow heads) (E). Scale bar represents 20 µm.</p

Fusion proteins VP22-hBcl-xL and hBcl-xL-VP22 failed to traffic between COS-1 cells and MSCs.

<p>COS-1 cells transfected with 0.5 µg phBcl-xL (A–D), pVP22-hBcl-xL (E–H) or phBcl-xL-VP22 (I–L) were trypsinized 24 hours after transfection and were co-plated with MSCs at a ratio of 1∶20. The co-cultured cell population were incubated for another 24 hours and were double stained with anti-Bcl-xL (B, F and J) and anti-T-antigen (C, G and K) antibodies. Bcl-xL was detected only in T-antigen positive COS-1 cells precluding the intercellular spread of VP22-hBcl-xL and hBcl-xL-VP22. Scale bar represents 20 µm.</p

Intercellular trafficking of VP22-myc between COS-1 cells and MSCs.

<p>COS-1 cells transfected with 0.5 µg pVP22-myc DNA were trypsinized 24 hours after transfection and were co-plated with untransfected HeLa cells (A–D), MSCs (E–H) or BrdU labeled MSCs (I–L) at a ratio of 1∶20 respectively. The co-cultured cell populations were incubated for another 24 hours and were stained with anti-myc (B, F and J), anti-T-antigen (C, G and K) and anti-BrdU (I) antibodies. VP22-myc was detected not only in T-antigen positive COS-1 cells but also in the T-antigen negative HeLa cells (arrows in D) and in the T-antigen negative and/or BrdU positive MSCs (arrows in H and L), demonstrating the spread of VP22 protein. Scale bar represents 20 µm.</p

The surface marker expression of BrdU labeled bone marrow MSCs.

<p>The expressions of selected surface markers of BrdU labeled MSCs were analyzed by flowcytometry. Mouse IgG1 was used as an isotype control (A); in bone marrow MSCs, CD29, CD44, and CD90 were highly expressed (B–D) while the markers for hematopoietic stem cells, CD34 and CD45, were not expressed (E and F).</p

The surface marker expression of rat bone marrow MSCs.

<p>The expressions of selected surface markers of P3 MSCs were analyzed by Flowcytometry. Mouse IgG1 was used as an isotype control (A); in bone marrow MSCs, CD29, CD44, and CD90 were highly expressed (B–D) while the markers for hematopoietic stem cells, CD34 and CD45, were not expressed (E and F).</p

Fusion proteins VP22-hBcl-xL and hBcl-xL-VP22 are present in the insoluble fractions of the cell lysates.

<p>Cos-1 cells were harvested 48 h after transfection and lysed either in RIPA lysis buffer or in NP40 lysis buffer. The cell lysates in NP-40 lysis buffer were further sonicated and centrifuged to obtain fractions containing soluble supernatants (Sup) and insoluble pellets (Pellet). Proteins were resolved by 10% SDS-PAGE and transferred to PVDF membranes, then probed with mouse anti-myc antibody (A), rabbit anti-Bcl-xL antibody (B) and mouse anti-β-Tubulin antibody (loading control). It was shown that at least one half of the total hBcl-xL protein was present in the soluble supernatants while VP22-hBcl-xL and hBcl-xL-VP22 were absent, indicating the solubility of Bcl-xL in the absence of VP22. And, instead, VP22-hBcl-xL and hBcl-xL-VP22 were detected in the insoluble pellets of the cell lysates, indicating the interactions of the fusion proteins with other cellular components which could preclude the intercellular trafficking of the fusion proteins.</p

VPA improves ferroptosis in tubular epithelial cells after cisplatin-induced acute kidney injury

Background: As a novel non-apoptotic cell death, ferroptosis has been reported to play a crucial role in acute kidney injury (AKI), especially cisplatin-induced AKI. Valproic acid (VPA), an inhibitor of histone deacetylase (HDAC) 1 and 2, is used as an antiepileptic drug. Consistent with our data, a few studies have demonstrated that VPA protects against kidney injury in several models, but the detailed mechanism remains unclear. Results: In this study, we found that VPA prevents against cisplatin-induced renal injury via regulating glutathione peroxidase 4 (GPX4) and inhibiting ferroptosis. Our results mainly indicated that ferroptosis presented in tubular epithelial cells of AKI humans and cisplatin-induced AKI mice. VPA or ferrostatin-1 (ferroptosis inhibitor, Fer-1) reduced cisplatin-induced AKI functionally and pathologically, which was characterized by reduced serum creatinine, blood urea nitrogen, and tissue damage in mice. Meanwhile, VPA or Fer-1 treatment in both in vivo and in vitro models, decreased cell death, lipid peroxidation, and expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), reversing downregulation of GPX4. In addition, our study in vitro indicated that GPX4 inhibition by siRNA significantly weakened the protective effect of VPA after cisplatin treatment. Conclusion: Ferroptosis plays an essential role in cisplatin-induced AKI and inhibiting ferroptosis through VPA to protect against renal injury is a viable treatment in cisplatin-induced AKI. </p

Risk factors associated with intraoperative hypothermia, Beijing, China (N = 830).

<p>OR, Odds Ratio, Significant level indicates as P<0.05.</p><p>¹ adjusted OR were presented after adjusting all the variables in above table.</p><p>² patients receiving unwarmed IV fluid only</p><p>Risk factors associated with intraoperative hypothermia, Beijing, China (N = 830).</p

Change of intraoperative core temperature during operations.

<p>Patients’ core temperature was measured at the tympanic membrane beginning every 15 minutes after the induction of anesthesia and until the end of the operation. A total of 830 subjects were enrolled in the study; 89 (10.7%) received active warming, and 741(89.3%) received no warming.</p