Fig 2: Expression of TRPM8 in tumor and non-tumor prostate cells. (A and B) Expression of TRPM8 in LNCaP, DU145 and PNT1A cells, detected by (A) RT-PCR and (B) western blot analysis. β-actin and GAPDH were used as positive controls in RT-PCR and western blot analysis, respectively. Water was used as a negative control in RT-PCR. (C and D) TRPM8 expression was quantified from (C) RT-PCR and (D) western blot analysis. LNCaP cells were used as positive controls for the expression of TRPM8. TRPM8, transient receptor potential melastatin 8; RT-PCR, reverse transcription-polymerase chain reaction.

Fig 3: CD38 deficiency impairs autophagy formation in LPS/GalN-induced acute liver injury. A) Cd38+/+ and Cd38−/− mice were intraperitoneally injected with LPS (2 μg/kg) and GalN (700 mg/kg; n = 5). After 6 h, the livers were fixed, sectioned, and stained with uranyl acetate and lead citrate. Autophagosomes were examined by transmission electron microscopy. Arrows indicate autophagosomes. Mit, mitochondria; Nu, nucleus. Magnification, ×1 μm. B) The number of autophagosomes was quantified for each experiment (n = 3) under the conditions described in A. Data are means ± sd. *P < 0.001. C) CD38 is involved in LPS-induced autophagy of hepatocytes. Primary hepatocytes prepared from Cd38+/+ and Cd38−/− mice were treated with 100 ng/ml LPS for the indicated time. Cells were lysed, and immunoblotting was performed to determine LC3 (autophagy marker), p62, ATG5, ATG7, Bcl-2, Caspase-3, Caspase-9, and GAPDH. D) The plot shows the means ± sd from 3 independent experiments. *P < 0.001 vs. control, **P < 0.01, Cd38+/+ vs. Cd38−/−. E) Comparison of liver apoptosis in Cd38+/+ and Cd38−/− mice 6 h after treatment with 2 μg/kg LPS and 700 mg/kg GalN using TUNEL staining and H&E stain. Scale bars, 50 μm.

Fig 4: β-Trcp binds to Mxi1 and promotes the ubiquitination and degradation of Mxi1. A. Mxi1-associated proteins in HEK293T cells identified by tandem affinity purification and mass spectrometry analysis were presented. The recovered peptide numbers for a given protein were listed as indicated. B. Exogenously expressed β-Trcp interacts with Mxi1 and vice versa. HEK293T cells were transfected with indicated constructs. Cell lysates were subjected to pulldown using S protein beads or immunoprecipitation (IP) using anti-Myc beads and then analyzed by Western blotting using indicated antibodies (n=3). C. Endogenous β-Trcp binds to Mxi1 and vice versa. HeLa cell lysates were subjected to immunopreciptation using IgG or anti-β-Trcp antibodies and then analyzed by Western blotting as indicated (n=3). D. β-Trcp knockdown leads to accumulation of endogenous Mxi1. HeLa and H1299 cells were transfected with control or β-Trcp siRNA for 48 h and then analyzed by Western blotting as indicated (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). E. Overexpression of β-Trcp1 decreases the levels of Mxi1 protein. HeLa cells were transfected with increasing amounts of β-Trcp1, followed by Western blotting with indicated antibodies (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). F. HeLa cells were transfected with indicated constructs for 24 h and treated with MG132 (10 μM) for another 4 h. The samples were subjected to immunopreciptation (IP) using anti-Flag beads and then analyzed by Western blotting using indicated antibodies (n=3). WCL: whole cell lysate. G. Upper panel: HeLa cells transfected with indicated siRNA for 48 h were treated with CHX (20 mg/mL) for the indicated times and then analyzed by Western blotting using indicated antibodies. Lower panel: Quantification of the Mxi1 band intensities over time (n=3). H. Alignment of the candidate phosphodegron sequence in Mxi1 from different species. I. HeLa cells stably expressing empty vector (EV), wild-type Mxi1, or Mxi1 S160A mutant were transfected with indicated constructs and then treated with MG132 (10 μM) for another 4 h. The samples were analyzed as described in (E) (n=3).

Fig 5: Expression and distribution of ZO-1 and Occludin proteins in mice colon tissue according to immunohistochemistry. (A) Immunohistochemistry detected the distribution of ZO-1 and occludin in colon tissue. Magnification, ×40. Eight fields were randomly collected under the microscope, and the percentage of positive cells and the intensity of staining were scored. **P<0.01, ***P<0.001. (B) Western blot assay detected the levels of ZO-1 and occludin in colon tissue. The results were normalized to GAPDH, and experiments were conducted at least three times. Data are presented as the mean ± standard deviation. ***P<0.001 vs. normal. ###P<0.001 vs. DSS. DSS, dextran sulfate sodium; SASP, sulfasalazine; ZO-1, zonular occludens-1.