Journal: BMC Developmental Biology

Article Title: Protein interactions of the transcription factor Hoxa1

doi: 10.1186/1471-213X-12-29

Figure Lengend Snippet: Bimolecular Fluorescence Complementation assay reveals the Hoxa1-PBX1A interaction in culture cells. COS7 cells were transfected with combinations of VN173, VN173-PBX1A, VC155 and VC155-Hoxa1 expression vectors. Upon interaction between PBX1A and Hoxa1, the VN173 and VC155 moieties of the Venus fluorescent protein brought together provide a fluorescent signal ( A ). Fluorescence complementation does not appear when the VN173 or VC155 fragments are expressed instead of the corresponding fusion proteins ( B - C ). As a control, expression of Venus fragments is detected by immunocytochemistry (anti-GFP). The BiFC signal shows colocalization with the anti-GFP immunofluorescence ( D - F ).

Article Snippet: Cells were further blocked with 10% low-fat milk in TBS-0.1% Triton X100 solution for 45 min at room temperature, followed by overnight incubation in TBS-0.1% Triton X100 solution at 4°C, with a rabbit polyclonal anti-GFP (Invitrogen A11122, diluted 1/200), a mouse anti-GST (Sigma G1160, diluted 1/50), a mouse monoclonal anti-TRAF1 (Santa Cruz, sc-6253, diluted 1/50), or a rabbit polyclonal anti-Hoxa1 (Abcam ab64941, diluted 1/50), as primary antibodies.

Techniques: Bimolecular Fluorescence Complementation Assay, Transfection, Expressing, Fluorescence, Immunocytochemistry, Immunofluorescence

Journal: Stem Cell Reports

Article Title: Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

doi: 10.1016/j.stemcr.2019.06.006

Figure Lengend Snippet: MYOD and PAX3 Are Not Sufficient to Induce Myogenic Stem Cells, but Result in Differentiated Cells (A) Schematic representation of skeletal muscle development in mouse embryogenesis. Pax3 GFP/ + ( Pax3-GFP ) indicates dermomyotome (DM; green), and MyoD-Cre ; Rosa26 CAG-LSL-tdTomato/ + ( MyoD-tdTomato ) primed populations were initially labeled in myotome (M; Red). NT, neural tube; DRG, dorsal root ganglia; N, notochord; DML, dorsomedial lip; VLL, ventrolateral lip. (B) Mouse Pax3 , MyoD , and Pax3 + MyoD infected mouse embryonic fibroblasts (MEFs) derived from Pax3-GFP ; MyoD-tdTomato embryos after 7 days. Scale bars, 50 μm. (C) FACS analyses with MEFs following the time course after MyoD infection, from day 3 to day 14.

Article Snippet: Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C.

Techniques: Labeling, Infection, Derivative Assay

Journal: Stem Cell Reports

Article Title: Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

doi: 10.1016/j.stemcr.2019.06.006

Figure Lengend Snippet: The Combination of Four Transcription Factors Induces PAX3-Expressing Myogenic Cells from Mouse Fibroblasts (A and B) Detectable GFP expression in MEFs (arrowheads) infected with eight transcription factors (+8F) after 14 days. (C) FACS analyses of GFP-expressing cells with mouse Pax3 , HeyL , and Klf4 (+3F) and persistent MYOD (++DOX) or transient MYOD for 72 h (+DOX). (D) Immunofluorescence for GFP (labeled with Alexa 488, green) and Pax7 (labeled with Alexa 647, red) with GFP-positive cells induced by 3F and transient MYOD acceleration (+3F+DOX). (E) Expression levels of Pax7 , Calcr , Spry1 , Sdc4 , Sox1 , and Sox10 transcripts levels in induced Pax3-GFP-positive (gray) or -negative (white) cells with 3F+DOX. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗∗ p < 0.01, ∗ p < 0.05. (F) Morphological features of cultured GFP-positive satellite cells (GFP + MuSCs; upper panel) and three transcription factors with transient DOX treatment (+3F+DOX GFP + cells; lower panel) for 4 days. (G) Myogenic transcripts of Pax7 , endogenous MyoD ( MyoD UTR ), and Myog relative to Rpl13a transcripts in GFP + cells cultured for 4 days. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.05. (H) Differentiated myogenic cells (DAPI, blue; MyHC, Alexa 647, red) from induced GFP-positive cells with 3F+DOX (right panels) compared with mouse Pax3-GFP satellite cells (GFP + MuSCs; left panels) on 2% horse serum for 7 days. (I) Quantification of the ratio of DAPI-positive mono (N = 1) or multiple nuclei (N = 2, 3 or N > 3) present in single MyHC-positive myofibers of (H). n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.05. (J) Transcriptional levels of myogenic markers Pax7 , MyoD , Myog , Myh3 , Myh7 , and Myh1 , differentiated for 7 days from GFP + cells. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.05. Error bars indicate ±SEM. Scale bars, 50 μm.

Article Snippet: Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C.

Techniques: Expressing, Infection, Immunofluorescence, Labeling, Two Tailed Test, Cell Culture

Journal: Stem Cell Reports

Article Title: Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

doi: 10.1016/j.stemcr.2019.06.006

Figure Lengend Snippet: Mesodermal Differentiation from PAX3 GFP/ + -Derived hiPSCs (A) GFP expression in induced teratoma (left panels) and neural precursors by SFEBq induction (right panels) derived from PAX3 GFP/ + hiPSCs. Scale bars, 200 μm. (B) Immunostaining with Pax3 antibody in GFP-sorted cells (upper panel) and the ratio of PAX3 transcript relative to RPL1 3A in GFP-sorted cells (lower panel). Red, PAX3; blue, DAPI. Scale bar, 100 μm. (C) FACS profiles of Pax3 GFP/ + ; Tet-On MYOD hiPSCs with or without continuous doxycycline (DOX) treatment (left panel, no DOX; middle panel, continuous DOX) and transient treatment for 72 h and cultured for a total of 7 days (right panel, DOX for 72 h). (D) Relative transcription levels of dermomyotome ( DMRT2 ), myogenic ( MYF5 ), and neural ( SOX1 , SOX10 ) progenitors in induced GFP-positive cells treated with doxycycline for 72 h and cultured for a total of 7 days. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.05. (E) Schematic representation of the induction to mesodermal cells from hiPSCs. (F) Morphological features and GFP expression of mesodermal differentiated cells derived from PAX3 GFP/ + ; Tet-On MYOD hiPSCs. (G) Cultured cell populations on mesodermal induction for 7 days (left panel) and subpopulation of ectodermal GFP (GFP + DLL1 − ) and mesodermal GFP (green box, GFP + DLL + ) derived from presomitic mesoderm (GFP − DLL1 + ) labeled with the hDLL1 antibody (right panel). (H) Transcription analyses of each type of sorted cells with presomitic mesodermal markers ( MSGN , TBX6 , PCGFRa ) and the dermomyotome marker DMRT2 . n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ∗ p < 0.05, ∗∗ p < 0.01. (I) Transcription analyses with PAX3-GFP-sorted cells on each differentiated condition. n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ∗ p < 0.05, ∗∗ p < 0.01. Error bars indicate ±SEM.

Article Snippet: Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C.

Techniques: Derivative Assay, Expressing, Immunostaining, Cell Culture, Two Tailed Test, Labeling, Marker

Journal: Stem Cell Reports

Article Title: Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

doi: 10.1016/j.stemcr.2019.06.006

Figure Lengend Snippet: Selected Four Transcription Factors Induce Human PAX3-Positive Muscle Stem Cells Derived from the Mesodermal Lineage (A) Procedure for the induction of PAX3-GFP cells derived from mesodermal lineage. (B) GFP or mCherry expression in transient MYOD-induced populations after expression of PAX3, HEYL, and KLF4 transcription factors (+3F), and doxycycline for 72 h (+DOX). (C) The contrasting proportion of induced PAX3-GFP cells with transient single doxycycline treatment (+DOX) and three transcription factors and transient/continuous doxycycline (+3F+DOX/+3F++DOX). (D) The FACS profile of CD56 and CD82 expression, human myogenic cell-surface markers, in induced cells. More than 60% of PAX3-GFP cells were merged with CD56 + CD82 + as double positives. (E) Immunostaining with anti-PAX7 (left panel, labeled with Alexa 488, light blue) and anti-MYOGENIN (MYOG; right panel, labeled with Alexa 647, purple) antibodies in CD56 + CD82 + GFP + sorted cells. GFP signals were not detected after immunostaining. (F and G) In vitro myogenic differentiation assay with induced PAX3-GFP-positive (CD56 + GFP + ) or -negative (CD56 + GFP − ) cells of CD56-positive cells in 2% of horse serum medium for 7 days. MYOG, purple; MYHC, light blue (labeled with Alexa 647); DAPI, blue. (H) Percentage of differentiated myogenic cells presented as the ratio of MYOG or MYHC/DAPI fluorescence. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.01. (I) Ratio of transcripts of PAX3 relative to RPL1 3A in myogenic differentiated cells from PAX3-GFP-positive (+) or -negative cells (−) of the CD56 + population. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.01. (J) Fetal ( NFIX ) and adult ( NAP1L5 ) transcriptional analyses with PAX3-GFP-positive or -negative cells of CD56-positive cells induced by transient DOX treatment plus three transcription factors (+3F+DOX). Mesodermal GFP-expressing cells, marked by DMRT2 (dermomyotome), were used as control (GFP + mesoderm). n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ∗ p < 0.01. (K) Transcriptional analyses with GFP-positive cells induced by transient DOX treatment plus three transcription factors (+3F+DOX). GFP-expressing cells with or without DOX were used as control (GFP + −DOX/+DOX). n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ∗ p < 0.01. Error bars indicate ±SEM. Scale bars, 50 μm.

Article Snippet: Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C.

Techniques: Derivative Assay, Expressing, Immunostaining, Labeling, In Vitro, Differentiation Assay, Fluorescence, Two Tailed Test

Journal: Stem Cell Reports

Article Title: Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

doi: 10.1016/j.stemcr.2019.06.006

Figure Lengend Snippet: The Effect of Removing PAX3 from the Candidate Four Transcription Factors for Inducing PAX3-Expressing Muscle Stem Cells from hiPSCs (A) FACS profile for detecting PAX3-GFP cells with or without PAX3 from mesodermal cells. (B) Transcriptional analyses with GFP-positive cells induced by transient DOX treatment plus three transcription factors (+3F+DOX) or two transcription factors (no PAX3, +2F+DOX). n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.01. (C) Myogenic differentiation with induced PAX3-GFP-positive cells in 2% of horse serum medium for 7 days. MYOG, purple (labeled with Alexa 647); DAPI, blue. (D) Percentage of differentiated myogenic cells presented as MYOG/DAPI ratio. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ∗ p < 0.01. Error bars indicate ±SEM.

Article Snippet: Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C.

Techniques: Expressing, Two Tailed Test, Labeling

Journal: Stem Cell Reports

Article Title: Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

doi: 10.1016/j.stemcr.2019.06.006

Figure Lengend Snippet: Induced Human PAX3-GFP Cells Have High Regenerative Capacity in Dystrophin-Deficient Mice (A) Flowchart for the transplantation into DMD − /y ;NSG mice with PAX3-GFP-positive cells derived from human iPSCs. (B) Comparison between the same number of GFP-positive and -negative cells (+GFP + cells, +GFP − cells) for transplantation. Immunostaining for dystrophin (DYS; labeled with Alexa 594, red), human nuclear lamin A/C (hLMNA; labeled with Alexa 488, green), laminin-a2 (LAMA2; labeled with Alexa 647, white), and DAPI (blue) on engrafted tibialis anterior (TA) muscle with 1.0 × 10 6 GFP-positive cells. (C) Quantification of total DYSTROPHIN-positive (DYS+) regenerative myofibers on the section transplanted with equal numbers of GFP + cells induced for 7 days as embryonic mesodermal cells (day 7), GFP-positive or -negative cells accelerated with four transcription factors (+3F+DOX cells), and culture for 4 days after sorting for GFP-positive cells induced by four transcription factors (cultured GFP + ) or by DOX treatment alone (+DOX). n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ∗ p < 0.01. (D) Immunostaining for PAX7 (DYS; labeled with Alexa 594, red), human nuclear lamin A/C (hLMNA; labeled with Alexa 488, green), laminin-a2 (LAMA2; labeled with Alexa 647, white), and DAPI (blue) on a section of engrafted TA with +3F+DOX-induced PAX3-positive cells. (E) The quantification of total PAX7;hLMNA double-positive cell numbers on a section of transplanted myofibers to contribute as MuSCs in vivo with cells as in (C). n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ∗ p < 0.01. (F) Immunodetection with MYOD antibody (labeled with Alexa 594, red) on GFP-transplanted sections. MYOD-positive cells derived from mouse host (white arrowheads) and transplanted cells (green arrowheads) in the position of muscle satellite cells (left panel) and muscle interstitium (right panel). (G) Ki67 staining on a section of engrafted TA with +3F+DOX-induced GFP cells. White arrowheads indicate Ki67-positive cells (labeled with Alexa 647). Red arrowheads have no Ki67 signals on regenerating myofibers labeled with DYSTROPHIN (DYS; labeled with Alexa 594, red) and human nuclear lamin A/C (hLMNA; labeled with Alexa 488, green). Error bars indicate ±SEM. Scale bars, 50 μm.

Article Snippet: Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C.

Techniques: Transplantation Assay, Derivative Assay, Immunostaining, Labeling, Cell Culture, In Vivo, Immunodetection, Staining