Journal: PLoS ONE

Article Title: ANKK1 is found in myogenic precursors and muscle fibers subtypes with glycolytic metabolism

doi: 10.1371/journal.pone.0197254

Figure Lengend Snippet: (A) Alignment analysis of predicted amino acid sequences of ANKK1 kinase domain in Mus Musculus (GenBank: AAI45079.1) and Homo sapiens (NP_848605.1) and identification of NLS and NES. Shading indicates 100% sequence identity. The kinase domain is underlined. Highly hydrophobic amino acids in NESs are marked in bold. (B) ANKK1 (α-STk2) and α-ACTININ immunostaining of C2C12 myoblasts in differentiation (24–48 h) treated with 2 ng/ml LMB 24 h before fixed. Scale bar: 25 μm. Images were taken from confocal optical sections that are representative for the group averages. (C) Quantification of ANKK1 fluorescence intensity (a.u) in nucleus (N = 3) and (D) fusion index (N = 3). (E) Western blot of subcellular C2C12 fractions using α–STk2 and α–MyoD. β-ACTIN was used as control and α-TUBULIN as cytosolic marker. (F) Western blot bands were quantified using the ImageJ 1.45v software and the relative expression was represented related to β-ACTIN value. p < 0.05: *; p < 0.01: **; p < 0.001: ***. a.u: arbitrary units; N: Nucleus; C: Cytoplasm.

Article Snippet: The following mouse monoclonal antibodies were used: α-beta III Tubulin as a neuronal marker (1:7000, Tubb3, Promega), α–embryonic Myosin heavy chain (eMyHC) as embryonic myoblasts marker (1:80, clone F1.652-s, Developmental Studies Hybridoma Bank, DSHB); α-alpha-Actinin as myotubes marker (1:200, A7732, Sigma-Aldrich); α-Pax7 as SCs marker (1:50, Pax7-c, DSHB); α-MyoD as committed myoblasts marker (1:200, sc-32758, Santa Cruz Biotechnology).

Techniques: Sequencing, Immunostaining, Fluorescence, Western Blot, Control, Marker, Software, Expressing

Journal: PLoS ONE

Article Title: ANKK1 is found in myogenic precursors and muscle fibers subtypes with glycolytic metabolism

doi: 10.1371/journal.pone.0197254

Figure Lengend Snippet: (A) Representative images for the group averages of IHC analysis of ANKK1, MyHC1 and MyHC2 in mice adult skeletal gastrocnemius serial sections and (B) colocalization of ANKK1 and MyHC2 in triceps and soleus muscles. The black asterisk indicates the same fiber. Scale bar: 50 μm. (C) Glycolysis induction in C2C12 myoblasts using HG and berberine during 24 h treatments. Oxygen consumption was assessed using a Clark-type oxygen electrode (N = 7) and lactic acid production was measured (N = 3). Relative oxygen and L-lactic acid production values are shown (±SD). (D) ANKK1 expression analysis by reverse transcriptase polymerase chain reaction (N = 3). Relative mRNA values (±SD) are shown. (E) Western blot of subcellular C1C12 fractions using α-STk2 (N = 4). β-ACTIN was used as control and α-TUBULIN as cytosolic marker. Western blot bands were quantified using the ImageJ 1.45v software and the relative expression was represented related to β-Actin value. p < 0.05: *; p < 0.01: **; p < 0.001: ***.N: nucleus; C: cytoplasm; RE: Relative Expression; Ber: berberine.

Article Snippet: The following mouse monoclonal antibodies were used: α-beta III Tubulin as a neuronal marker (1:7000, Tubb3, Promega), α–embryonic Myosin heavy chain (eMyHC) as embryonic myoblasts marker (1:80, clone F1.652-s, Developmental Studies Hybridoma Bank, DSHB); α-alpha-Actinin as myotubes marker (1:200, A7732, Sigma-Aldrich); α-Pax7 as SCs marker (1:50, Pax7-c, DSHB); α-MyoD as committed myoblasts marker (1:200, sc-32758, Santa Cruz Biotechnology).

Techniques: Muscles, Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Control, Marker, Software

Journal: PLoS ONE

Article Title: ANKK1 is found in myogenic precursors and muscle fibers subtypes with glycolytic metabolism

doi: 10.1371/journal.pone.0197254

Figure Lengend Snippet: (A) ANKK1 (α-STk2) and ACTININ immunostaining of C2C12 cells during myogenic differentiation. D0: proliferative myoblast; D3: myoblasts in differentiation; D6: myotubes. (B) Quantification of nuclear ANKK1 fluorescence intensity (a.u.) (N = 3) (C) Western blot analysis of C2C12 subcellular fractions. β-ACTIN was used as control and α-LAMIN A/C as nuclear marker (N = 1). (D) Rhabdomyosarcoma (RD) myoblasts differentiation, (E) quantification of nuclear ANKK1 (N = 3) and (F) Western blot of subcellular fractions (N = 1). (G) Immunostaining of ANKK1 with PAX7 and MYOD in C2C12 cells, (left) and quantification of positive nuclei for ANKK1, PAX7 and MYOD along myogenic differentiation (right, N = 3). Images were taken from confocal optical sections that are representative for the group averages. Scale bar: 25 μm p < 0.05: *; p < 0.01: **; p < 0.001: ***. D: Days after induction of differentiation; a.u: arbitrary units; N: Nucleus; C: Cytoplasm.

Article Snippet: The following mouse monoclonal antibodies were used: α-beta III Tubulin as a neuronal marker (1:7000, Tubb3, Promega), α–embryonic Myosin heavy chain (eMyHC) as embryonic myoblasts marker (1:80, clone F1.652-s, Developmental Studies Hybridoma Bank, DSHB); α-alpha-Actinin as myotubes marker (1:200, A7732, Sigma-Aldrich); α-Pax7 as SCs marker (1:50, Pax7-c, DSHB); α-MyoD as committed myoblasts marker (1:200, sc-32758, Santa Cruz Biotechnology).

Techniques: Immunostaining, Fluorescence, Western Blot, Control, Marker

Journal: PLoS ONE

Article Title: ANKK1 is found in myogenic precursors and muscle fibers subtypes with glycolytic metabolism

doi: 10.1371/journal.pone.0197254

Figure Lengend Snippet: (A) Alignment analysis of predicted amino acid sequences of ANKK1 kinase domain in Mus Musculus (GenBank: AAI45079.1) and Homo sapiens (NP_848605.1) and identification of NLS and NES. Shading indicates 100% sequence identity. The kinase domain is underlined. Highly hydrophobic amino acids in NESs are marked in bold. (B) ANKK1 (α-STk2) and α-ACTININ immunostaining of C2C12 myoblasts in differentiation (24–48 h) treated with 2 ng/ml LMB 24 h before fixed. Scale bar: 25 μm. Images were taken from confocal optical sections that are representative for the group averages. (C) Quantification of ANKK1 fluorescence intensity (a.u) in nucleus (N = 3) and (D) fusion index (N = 3). (E) Western blot of subcellular C2C12 fractions using α–STk2 and α–MyoD. β-ACTIN was used as control and α-TUBULIN as cytosolic marker. (F) Western blot bands were quantified using the ImageJ 1.45v software and the relative expression was represented related to β-ACTIN value. p < 0.05: *; p < 0.01: **; p < 0.001: ***. a.u: arbitrary units; N: Nucleus; C: Cytoplasm.

Article Snippet: The following mouse monoclonal antibodies were used: α-beta III Tubulin as a neuronal marker (1:7000, Tubb3, Promega), α–embryonic Myosin heavy chain (eMyHC) as embryonic myoblasts marker (1:80, clone F1.652-s, Developmental Studies Hybridoma Bank, DSHB); α-alpha-Actinin as myotubes marker (1:200, A7732, Sigma-Aldrich); α-Pax7 as SCs marker (1:50, Pax7-c, DSHB); α-MyoD as committed myoblasts marker (1:200, sc-32758, Santa Cruz Biotechnology).

Techniques: Sequencing, Immunostaining, Fluorescence, Western Blot, Control, Marker, Software, Expressing