Additional file 3: Figure S2. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

H&E-stained histological sections of the gastrocnemius and quadriceps from 8-month-old mice of the indicated genotypes. Scale bar = 50 μm. The number of mice is 6–8 for each group

Additional file 4: Figure S3. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Masson’s trichrome-stained histological sections of the gastrocnemius and quadriceps from 8- to 18-month-old mice of the indicated genotypes. Scale bar = 50 μm. The number of mice is 6–8 for each group

Additional file 9: Table S4. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Gene list. List of genes that are upregulated in the KO skeletal muscles

Additional file 2: Figure S1. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Test of anti-Ano5 antibodies by Western blotting. a The anti-Ano5 antibody (sc-169626) from Santa Cruz Biotechnology did not detect any positive signal in human Ano5-expressing cell lysate, which was correctly detected by the anti-GFP antibody at the predicted size. b The anti-Ano5 antibody (AP8580B) from Abgent produced a prominent band at about 100 kDa in both negative and positive samples; however, this band is not Ano5 because the Ano5-YFP fusion protein should be around 135 kDa as correctly detected by the anti-GFP antibody. The arrows point to the correct Ano5-YFP fusion protein. These experiments were repeated at least three times

Additional file 6: Figure S5. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Representative images of Masson’s trichrome-stained histological sections of WT and Ano5 KO hearts after PBS or ISO treatments for 2 weeks. Scale bar = 50 μm. The number of mice (18 months of age) is 6–8 for each group

Additional file 8: Table S3. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Gene list. List of genes that are downregulated in the KO skeletal muscles

Additional file 7: Table S2. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Echocardiographic measurements. Echocardiographic measurements in WT and Ano5 KO mice after isoproterenol injections

Hypoxia itself modulates BMMSC signaling pathways.

<p>Reverse phase protein arrays were performed under either normoxic or hypoxic conditions. <b>Panel A</b>: RPPA graph of two sets of untreated BMMSCs grown under normoxic conditions or low O₂ tension. When cells were 80% confluent they were switched to serum-free media for 3 h. Then cells were washed twice with PBS and lysis buffer was added to the plates. Results are expressed as means ±SEM for three independent experiments. *<i>p</i>≤0.05 and # <i>p</i>≤0.01. Percent change of untreated BMMSCs in hypoxia was determined as percent change of untreated BMMSCs in normoxia (control = 100%). RPS6: Ribosomal protein s6 (S6_pS240_S244-R-V). CAV1: Caveolin 1 (Caveolin-1-R-V). STAT3: Signal transducer and activator of transcription 3 (STAT3_pY705-R-V). AKT1 AKT2 AKT3: protein kinase B (Akt_pT308-R-V). CTNNB1: β-catenin (beta-Catenin-R-V). NOTCH1: Notch homolog 1 (Notch1-R-V). YBX1: The Y-box-binding Protein (YB-1-R-V). NRAS: Neuroblastoma RAS viral (v-ras) oncogene (N-Ras-M-V). MAP2K1: Mitogen activated protein kinase (MEK1-R-V). EGFR: Epidermal growth factor receptor (EGFR_pY1173-R-V). G6PD: Glucose-6 phosphate dehydrogenase (G6PD-M-V). EIF4EBP1: Eukaryotic initiating factor 4 (4E-BP1-R-V). CDKN1B: Cyclin dependent kinase inhibitor (p27_pT198-R-V). EIF4EBP1: Eukaryotic initiating factor 4 (4E-BP1_pS65-R-V). MAPK8: c-Jun N-terminal kinases (JNK_pT183_pT185-R-V). TRFC: Total rosette forming cells CD71 marker (TRFC-R-V). PRKAA1: 5'-AMP-activated protein kinase catalytic subunit alpha-1 (AMPK_pT172-R-V). <b>Panel B</b>: RPPA heatmap showing up-regulation of Akt (more than one antibody), CAV1, STAT3 and NOTCH1 in untreated BMMSCs in hypoxia vs. untreated BMMSCs in normoxia (control).</p

Modulatory effects of calcium on Tryptophan stimulated ERK phosphorylation.

<p>Erk (phospho Erk/Total Erk) assesed by Western blots of BMMSCs grown under normoxic conditions (21% O₂/5% CO₂). When cells were 80% confluent, they were treated with either Trp alone (100 µM) or in combination with either additional extracellular calcium (baseline calcium: 1.2 mM increased to 1.8 mM) or a calcium receptor antagonist (NPS 10 nM) in serum-free media. The cells were washed twice with PBS and lysis buffer was added to the plates. Results are expressed as means ± SEM for at least three independent experiments. *<i>p</i>≤0.05 and # <i>p</i>≤0.01.</p

Aromatic amino acid stimulated effects on signaling pathways under normoxic vs hypoxic conditions.

<p>Reverse-phase protein array graphs of two sets of BMMSCs grown under normoxic conditions (21% O₂/5% CO₂) and under low O₂ tension (3% O₂/5% CO₂; physiologic hypoxia). When cells were 80% confluent they were treated with Phe, Tyr or Trp (100 µM) in serum-free media for 3 h. The cells were washed twice with PBS and lysis buffer was added to the plates. a-Phe, b-Tyr and c-Trp. Results are expressed as means ± SEM for three independent experiments. *<i>p</i>≤0.05 and # <i>p</i>≤0.01. Percent change of hypoxia over control for Phe was determined as percent change of BMMSCs treated with Phe under hypoxic conditions (3% O₂%) vs. BMMSCs treated with Phe under normoxic conditions (21% O₂) (control = 100%). Both sets of BMMSCs were treated with Phe and grown under different oxygen levels. Same for Tyr and Trp pannels. RPS6: Ribosomal protein s6 (S6_pS240_S244-R-V). CAV1: Caveolin 1 (Caveolin-1-R-V). NDRG1: N-myc downregulated gene (NDRG1_pT346-R-V). AKT1 AKT2 AKT3: protein kinase B (Akt_pT308-R-V). STAT3: Signal transducer and activator of transcription 3 (STAT3_pY705-R-V). CTNNB1: β-catenin (beta-Catenin-R-V). NOTCH1: Notch homolog 1 (Notch1-R-V). MAP2K1: Mitogen activated protein kinase (MEK1-R-V). EIF4EBP1: Eukaryotic initiating factor 4 (4E-BP1_pT37_T46-R-V). MAPK8: c-Jun N-terminal kinases (JNK_pT183_pT185-R-V). LCK: Lymphocyte-specific protein tyrosine kinase (Lck-R-V). CCNE1:Cyclin E1 (Cyclin_E1-M-V). BCl2: B-cell lymphoma 2 (Bcl-2-M-V). PIK3R1: Phosphatidylinositol 3-kinase regulatory subunit alpha (PI3K-p85-R-V). PKC: Protein kinase C (PKC-pan_BetaII_pS660-R-V). C12ORF5: TP53-inducible glycolysis and apoptosis regulator (TIGAR-R-V). PEA15: Phosphoprotein Enriched in Astrocytes 15 (PEA15_pS116-R-V). TGM2: transglutaminase2, C polypeptide, protein-glutamine-gamma-glutamyltransferase(Transglutaminase-M-V).</p