Fig 1: 3D model of the mutant Cav3.2 channel reveals significant structural changes.A Predicted topologies of the wild type (WT) and mutant (MUT) Cav3.2 channels showing the four homologous domains (I–IV) in different colors, each composed of six transmembrane segments (bars S1–S6). The 52-amino acid insertion (yellow) is located between segments S5 and S6 of domain II (between residues Q969 and I970). The hydropathy profiles of the amino acid sequences are shown below the channel topologies. B Structural features of the WT and MUT Cav3.2 channels. Bi Simulations of the proteins viewed from the cytosolic side of the plasma membrane are shown in cartoon representation and colored by domain (DI–DIV). Domain II is colored in magenta and the 52-amino acid insertion is detached in yellow. Bii Structural view of the pore center showing the differences in the pore diameter between WT and MUT Cav3.2 channels.
Fig 2: Propofol alleviates H/R-induced neurological impairment by decreasing Cx43 and Cav3.2. Rat middle cerebral artery occlusion (MCAO) model was generated and animals were treated with/without propofol and intraventricularly injected with negative control (NC) or Cx43 overexpression lentivirus (Cx 43-OE). Sham group is used as the control here. (A) Neurobehavioral analysis is performed and scores are assessed. (B) TTC staining is used to assess the cerebral infarction, and the infarct volume was analyzed. (C) Apoptosis was measured using TUNEL staining and the protein expression and localization of Cx43, Cav3.2, Iba1, and Map2 was analyzed by immunofluorescence staining. *p < 0.05, **p < 0.01 vs. Sham. #p < 0.05, ##p < 0.01 vs. MCAO. n = 3.
Fig 3: Expression levels of TRPC3, Cav1.2, Cav.3.1 and Cav3.2 in human myometrial smooth muscle cells derived from the non-pregnant, full-term without labor onset, preterm, and full-term with labor onset patient groups (n=20/group). (A) Western blot analysis of protein expression levels in the different groups. GAPDH was used as the loading control. Quantified western blot analyses of relative expression levels of (B) TRPC3, (C) Cav1.2, (D) Cav.3.1 and (E) Cav3.2. Data are presented as the mean ± standard error. *P<0.05 vs. non-pregnant group. TRPC3, canonical transient receptor potential 3.
Fig 4: Mutant Cav3.2-mediated hyperfunction of mTORC1 signaling acts alone to cause enhanced proliferation of F2688-1-derived NPCs and together with impaired Reelin signaling in the abnormal migration of these cells.A Representative phase-contrast microscopy photographs of control- and F2688-1-derived NPCs. The bar graph shows the median cell body size (including soma size and all cell surface projections) of control-derived NPCs (n = 4) and F2688-1-derived NPCs (NPCs derived from 2 iPSC clones). There were no significant differences in median body size between the groups. B Line graph showing cell proliferation curves (live cell numbers at Day 0, 48 h, 72 h) of control-derived NPCs (n = 4) and F2688-1-derived (NPCs derived from 2 iPSC clones) cultured in the presence of either vehicle, 1 µM of NNC 55-0396 (NNC), or 100 nM of rapamycin; and in the presence of either mock-conditioned medium (mock) or Reelin-conditioned medium (Reelin). The graph shows the median value and interquartile range for each group. F2688-1 NPCs show significantly higher proliferation rates compared with control NPCs, which were completely rescued by treatment with either rapamycin or NNC. On the other hand, treatment with Reelin had no effect on the proliferation of F2688-1 NPCs. C Line graph showing the percentage relative wound density (RWD) over time in control-derived NPCs (n = 4) and in F2688-1-derived NPCs (NPCs derived from 2 iPSC clones) cultured in the presence of either vehicle, 1 µM of NNC, or 100 nM of rapamycin; and in the presence of either mock or Reelin. The graph shows the median value and interquartile range for each group. F2688-1 NPCs exhibit significantly higher migration rates compared with control NPCs, which was significantly attenuated by treatment with rapamycin, and completely rescued to levels equal to those of the untreated control NPCs in the exponential phase of the healing curve by treatment with either NNC or Reelin (analyzed time interval: 15 h–25 h). **p = 0.01, ****p = 0.0001.
Fig 5: Expression levels of TRPC3, MAPK, P-ERK, Cav1.2, Cav.3.1 and Cav3.2 in mouse myometrial smooth muscle cells derived from the non-pregnant, preterm, infected preterm and full-term groups (n=10/group). (A) Western blot analysis of protein expression levels in the different groups. GAPDH was used as the loading control. Quantified protein expression levels of (B) TRPC3, (C) MAPK, (D) P-ERK, (E) Cav1.2, (F) Cav.3.1 and (G) Cav3.2. Data are presented as the mean ± standard error. *P<0.05 vs. non-pregnant group. TRPC3, canonical transient receptor potential 3; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; P-, phosphorylated.
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