Fig 1: Autophagy reactivation inhibits Activin-induced chondrogenesis in FOP cells.A Chondrogenic differentiation micromass assay in ATDC5 ALK2R206H cells and in ATDC5 ALK2WT cells. Cells were incubated for 21 days in differentiation medium containing or not (NT), activin A (100 ng/ml) and treated, as indicated, with autophagy inhibitors Chloroquine (CQ, 20 μM), 3-Methyladenine (3-MA, 20 μM) or autophagy inducers Rapamycin (Rapa, 100 ng/ml), Spermidine (SPD, 20 μM) or DMSO. Representative images of Alcian blue staining. Histograms representing Alcian blue quantification measured by absorbance at 595 nm after solubilization with guanidine hydrochloride. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (**P ≤ 0.01; ***P < 0.001 ****P ≤ 0.0001). B Real-time PCR of Col10a1 mRNA in ATDC5 cells treated or not with activin A for 21 days in micromass cultures. mRplp0 was used to normalize data. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: unpaired t-test (**P ≤ 0.01; ***P < 0.001 ****P ≤ 0.0001). C Histogram showing measurement of ALP activity in ATDC5 cells treated or not as indicated for 5 days in micromass culture. 5 × 104 cells were homogenized in 1 ml of assay Buffer, diluted 1:10 in assay Buffer, and 80 μl was used to measure ALP activity. Assays were performed following the fluorimetric kit protocol (MAK411, Sigma–Aldrich) and measured at O.D. 405 nm reading with Varioskan LUX Plate Reader. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: A one-way ANOVA (Tukey’s multiple comparison) test was performed (**P ≤ 0.01; ***P < 0.001). Chondrogenic differentiation micromass assay in ATDC5 ALK2R206H cells stable expressing shRNA for ATG4 (shATG4) (D) or ATDC5 ALK2WT and ALK2R206H cells stable expressing shRNA for Rubicon (shRubicon) (E), upon lentiviral infection. A lentiviral empty vector was used as control (shCTRL). Cells were incubated for 21 days in differentiation medium containing or not (NT), activin A (100 ng/ml) and treated as indicated with Rapamycin (Rapa 100 ng/ml) or DMSO. Representative images of Alcian blue staining and relative histograms showing Alcian blue quantification measured by absorbance at 595 nm after solubilization with guanidine hydrochloride. All results represented the mean ± SD of at least three independent experiments for (D) and from six independent experiments for (E). Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (*P ≤ 0.05; **P ≤ 0.01, ****P ≤ 0.0001).
Fig 2: Autophagy is impaired in cells derived from FOP patients, and endogenous ALK2R206H receptor is degraded upon autophagy reactivation.A Representative immunoblotting and densitometric analysis with the indicated antibodies of Endothelial colony-forming cells (ECFCs) derived from FOP patients (FOP) carrying the R206H mutation or from healthy donors (CON) maintained in serum starvation conditions for 16 h and treated or not with CQ (20 µM, 2 h). B Representative immunoblotting and densitometric analysis with the indicated antibodies of total protein extracts from ECFCs cells derived from FOP patient in serum starvation conditions treated or not with Spermidine (SPD, 10 µM) or Rapamycin (Rapa,100 ng/ml) for 16 h. Histograms show the mean ± SD of at least three independent experiments. Statistical analysis: unpaired t-test (ns = not significant; *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001 ****P ≤ 0.0001).
Fig 3: Degradation of ALK2 by autophagy is partially impaired by the p.R206H mutation in hypoxic conditions.A Histograms show the mRNA expression for the indicated genes expressed as fold increase (mean ± SD) from three independent experiments in ATDC5 cells overexpressing ALK2WT or mutant ALK2R206H in normoxic (21% O2) and hypoxic conditions (1% O2,16 h). GAPDH mRNA was used to normalize data. B Immunoblotting and corresponding densitometric analysis with the indicated antibodies of ATDC5 cells overexpressing ALK2WT or mutant ALK2R206H in normoxic (21% O2) and hypoxic conditions (1% O2,16 h) (C) Histograms show the mRNA expression for the indicated gene expressed as fold increase (mean ± SD) from three independent experiments in ATDC5 cells overexpressing ALK2WT or mutant ALK2R206H in normoxic (21% O2) and hypoxic conditions (1% O2,16 h). GAPDH mRNA was used to normalize data. D, E Immunoblotting and relative densitometric analysis with the indicated antibodies of U2OS cells overexpressing ALK2WT or mutant ALK2R206H DDK tagged, in normoxic (21% O2) and hypoxic conditions (1% O2,16 h) and treated, as indicated, with 20 μM CQ (2 h). F Confocal microscopy analysis using anti-DDK (red) in U2OS cells expressing ALK2WT or mutant ALK2R206H DDK tagged receptors treated or not, as indicated, with chloroquine (CQ, 20 μM, 2 h) in normoxic (21% O2) and hypoxic conditions (1% O2,16 h). DNA was counterstained with DAPI (blue). Scale bar 10 µm. DDK fluorescence intensity was measured by using Fiji software (ImageJ). At least 50 cells from 3 independent experiments were analyzed unpaired t-test (Welch’s correction) was performed. Data are presented as mean ± SD of at least three independent experiments. Symbols (dots) represent individual cells. Statistical analysis: unpaired (ns = not significant; *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001 ****P ≤ 0.0001).
Fig 4: The ALK2 receptor is an autophagy target.A Representative images and relative quantification of EGFP-LC3-labeled autophagosomes colocalization with ALK2-DDK receptor. U2OS EGFP-LC3 cells expressing ALK2WT or mutant ALK2R206H DDK tagged receptors were treated with chloroquine (CQ, 20 μM, 2 h) in hypoxic conditions (1% O2,16 h). Images were acquired by spinning disk confocal microscopy and analyzed for colocalization of EGFP-LC3 (green) and DDK (red) dots using the ComeDet plugin of the Fiji ImageJ software. Data are presented as mean ± SD and normalized on total EGFP-LC3 dots. DNA was counterstained with DAPI (blue). Insets show a 3-fold enlargement of the boxed areas. Scale bar, 10 μm is shown. At least 50 cells from 3 independent experiments were analyzed unpaired t-test (Welch’s correction) was performed. (ns = not significant; *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001 ****P ≤ 0.0001). B Immunoblotting and relative densitometric analysis, numbers report the densitometric values of band intensity, with the indicated antibodies of U2OS cells overexpressing ALK2WT or mutant ALK2R206H in normoxic (21% O2) and hypoxic conditions (1% O2,16 h) and stably infected with lentiviral construct expressing shATG5 RNA interference or expressing an empty vector as control (shCTRL).
Supplier Page from OriGene Technologies for Activin Receptor Type IA (ACVR1) (NM_001105) Human Mutant ORF Clone