Fig 1: Macrophage-derived p47phox is not involved in antiparasitic defense during T. gondii infection. (A–C) BMDMs from p47phox +/+ and p47phox -/- mice were infected with GFP-RH strain (moi = 1) for the indicated time periods. (A) Cells were fixed and stained with Texas Red®-X phalloidin for labeling F-actin (red) for cytosolic fraction, and DAPI (blue) for nuclei and then analyzed for the number of GFP-RH strain using confocal microscopy (B,C) The number of GFP-RH strain per vacuole (for B) or of GFP-RH strain-infected cell (for C) were analyzed. Scale bar = 25 µm. Data are representative of five independent experiments (D,E) BMDMs from p47phox +/+ and p47phox -/- mice were infected with T. gondii RH strain (moi = 1) for the indicated time periods. (D) The mRNA expression for Mif and actb was determined using semiquantitative RT-PCR (top) or qPCR (bottom) analysis. (E) Immunoblot analysis was performed to determine protein expression of MIF and ß-tubulin. Data are representative of three independent experiments and are presented as means ± SD.
Fig 2: Nox4 is required for the host resistance against both RH and ME49 strain of T. gondii. (A) Survival of Nox4 +/+ and Nox4 -/- mice (n = 13 per genotype) infected with 200 tachyzoites of T. gondii RH strain (i.p. injection). (B–D) Nox4 +/+ and Nox4 -/- mice (n = 5 per genotype) were infected with 40 cysts of T. gondii ME49 strain (i.p. injection) for 20 days. (B) Number of cysts in brain of Nox4 +/+ and Nox4 -/- mice were counted using a microscope. (C) The mRNA expression for T. gondii-specific gene Sag1 in brain of Nox4 +/+ and Nox4 -/- mice was evaluated by qPCR analysis. (D) Serum MIF levels from Nox4 +/+ and Nox4 -/- mice were assessed by ELISA analysis. *P < 0.05, **P < 0.01, ***P < 0.001, compared with Nox4 +/+ mice infected with T. gondii (log-rank test (for A) or two-tailed Student’s t-test (for B–D).
Fig 3: Hypoxia Increased Chondrogenic Differentiation and MIF Expression while Decreasing Osteogenic Differentiation by HIF1ACESCs were induced under normoxia + PBS (NP), normoxia + DMOG (ND), hypoxia + PBS (HP), and hypoxia + YC1 (HY) conditions in chondrogenic induction medium (CIM) (A–C) or osteogenic induction medium (OIM) (D–G), respectively, for 3 weeks.(A) Chondrogenic gene (SOX9 and COL2), HIF1A, and MIF gene expression were assessed by RT-PCR of mRNA from CESCs induced in CIM.(B and C) Western blot analysis of the expression of SOX9, COL2, HIF1A, and MIF in samples treated under the conditions in (A). The protein contents were normalized according to ACTB level.(D) Osteogenic gene (RUNX2 and COL1), HIF1A, and MIF gene expression were assessed by RT-PCR of mRNA from CESCs induced in OIM.(E and F) Western blot analysis of the expression of RUNX2, COL1, HIF1A, and MIF in samples treated under the conditions in (D). The protein contents were normalized according to ACTB level.(G) Macrographs of alizarin red staining and ALP staining of CESCs treated under the conditions in (D).Data represent the mean ± SD (n = 3 independent experiments, t test). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: HIF1A Regulated Chondro-Osteogenic Differentiation of CESCs by MIFCESCs assigned to normoxia + PBS (NP) and hypoxia + YC1 (HY) groups, in which MIF expression should have been relatively low, were transfected with MIF-overexpressing lentiviral vector system (M+) and its scramble control (Scr). CESCs assigned to normoxia + DMOG (ND) and hypoxia + PBS (HP) groups in which MIF expression should had been relatively high were transfected with MIF-shRNA (M-) and its scramble control (Scr).(A) SOX9, COL2, HIF1A, and MIF gene expression were assessed by RT-PCR of mRNA from CESCs induced in CIM.(B and C) Western blot analysis of the expression of SOX9, COL2, HIF1A, and MIF in samples treated under the conditions in (A).(D) RUNX2, COL1, HIF1A, and MIF gene expression were assessed by RT-PCR of mRNA from CESCs induced in OIM.(E and F) Western blot analysis of the expression of RUNX2, COL1, HIF1A, and MIF in the samples treated under the conditions in (D).(G) Macrographs of alizarin red staining and ALP staining of CESCs treated under the conditions in (D).Data represent the mean ± SD (n = 3 independent experiments, t test). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: miR-451a functional studiesA. miRNA-451a expression by qRT-PCR in the PTC-derived cell lines TPC1, NIM1, K1 and BCPAP, and in the normal thyroid control cells T686. Data are presented relative to the value of T686 cells. B. MIF protein expression by western blot analysis (WB) in the same panel of cell lines; Actin is shown as loading control. Below the corresponding densitometric quantification; MIF expression was normalized to Actin and presented relative to the level of T686 cells. C. Relative expression of the pair miR-451a and MIF; data are derived from the analyses reported in (A) and (B). D-I. Functional studies in NIM1 and TPC1 cells either transfected with miR-451a synthetic mimic (miR-451a) or Negative-Control (NC). D, G. Representative images of transfected cells (LEICA inverted microscope, scale bar 100µm). Cell number was determined by nuclei staining; data are presented relative to NC-transfected cells. E, H. Cell proliferation following transfection. F, I. WB analysis (day 3 after transfection). Protein expression was quantified and normalized to the loading control GAPDH. Data are presented relative to NC-transfected cells. Graphs report mean ± s.e.m. of at least two independent experiments. * p-value<0.05, ** p-value<0.005, *** p-value<0.0001 by Student's t-test.
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