Journal: Molecular Medicine Reports

Article Title: PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population

doi: 10.3892/mmr.2019.9989

Figure Lengend Snippet: Pedigrees and sequence electropherograms of PITX3 mutations in 3 Chinese families with autosomal dominant congenital cataract. (A) The slit lamp photo of the right eye of the proband in Family 10003. Opacities were exhibited predominantly in the posterior capsule. (B) Pedigrees and sequence electropherograms of PITX3 mutations identified in 3 Chinese CC families. Family 10003 is a four-generation family with PSC. Families 10094 and 10178 are three-generation CC families. Individuals who underwent whole-exome sequencing are marked with triangle and individuals with available DNA samples were marked with asterisk. Probands are indicated by arrow in each pedigree. The black filled shapes signify individuals with cataract conditions, and the oblique line denotes the individuals who have succumbed. The question mark represents the individual identified to carry an asymptomatic mutation. Vertical arrows indicate the mutations in the index patients, respectively. (C) Schematic representation of PITX3 WT and mutated proteins. The green box represents the homeodomain of 60 amino acids and the blue box is the OAR domain of 14 amino acids. The aberrant protein segments caused by mutations are highlighted by the red box. CC, congenital cataract; M, mutant allele; +/WT, wild-type allele; OAR, homeobox protein orthopedia, Aristaless related homeobox and Retinal homeobox protein Rx domain.

Article Snippet: The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C.

Techniques: Sequencing, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population

doi: 10.3892/mmr.2019.9989

Figure Lengend Snippet: Functional analysis of the expression of PITX3 WT and mutants including. Subcellular localization and transactivation activity. (A) Subcellular localization of the PITX3 WT and mutant proteins transfected in HeLa cells. Cells were stained with PITX3 (N-20) primary antibody and Alexa Fluor 568 donkey anti-mouse IgG as a secondary antibody (red); DAPI was used as a nuclear counterstain (blue). Red fluorescence was not observed in HeLa cells without exogenous gene introduction. For cells transfected with PITX3 wild-type and mutants plasmids, the red fluorescence was localized predominantly in the nucleus. Western blot analysis indicated that the protein expression of PITX3 mutants was not affected. (B) Luciferase assay results for PITX3 WT and mutants co-transfected with the (C) pGL3-MIP (+58/-598), (D) pGL3-FOXE3 (−2988/-3722) or (E) pGL3-LEMD2 (−77/-985) reporters in 293T cells. All luciferase activities were normalized to β-galactosidase activity. In comparison with the empty vector pcDNA3.1, the values are indicated as fold changes of luciferase activity. *P≤0.05, **P≤0.01 and ***P≤0.001. PITX3, paired like homeodomain 3; WT, wild-type; MIP, lens fiber major intrinsic protein; FOXE3, forkhead box protein E3; LEMD2, LEM domain-containing protein 2; ns, not significant.

Article Snippet: The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C.

Techniques: Functional Assay, Expressing, Activity Assay, Mutagenesis, Transfection, Staining, Fluorescence, Western Blot, Luciferase, Comparison, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population

doi: 10.3892/mmr.2019.9989

Figure Lengend Snippet: Summary of 9 different mutations in PITX3 associated with cataract.

Article Snippet: The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C.

Techniques:

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: Vector map and sequencing results of the interference plasmid. (A) Vector GV102 map for constructing a plasmid carrying a sequence for Pitx3 interference. (B) Sequencing peak maps of sequences for Pitx3 interference. The upper, middle, and lower maps represent Sh-3568, Sh-3569, and Sh-3570 sequences, respectively.

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: Plasmid Preparation, Sequencing

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: Screening interference plasmids. (A) Cellular fluorescence 48 hours after transient transfection. Green cells indicate successful transfection of the plasmids. Scale bars: 50 μm. (B) Western blots for knockdown validation. The bars represent the ratio of optical density values of Pitx3 to β-actin. There was significant difference between the two groups ( P < 0.01, mean ± SD, n = 3, one-way analysis of variance and the least significant difference test). Control indicates the empty plasmid; Sh-3568, the Pitx3-Sh-3568 plasmid; Sh-3569, the Pitx3-Sh-3569 plasmid; and Sh-3570, the Pitx3-Sh-3570 plasmid, which were all transiently transfected into MES23.5 cells.

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: Fluorescence, Transfection, Western Blot, Plasmid Preparation

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: Effects of Pitx3 gene interference on glial cell line-derived neurotrophic factor (GDNF) transcriptional activity. GDNF-luciferase represents the cells transfected with the GDNF-luciferase plasmid alone. GDNF-luciferase + Pitx3-Sh-3570 represents the cells cotransfected with Pitx3-Sh-3570 and GDNF-luciferase plasmids. Transcriptional activity of GDNF decreased in the cells cotransfected with Pitx3-Sh-3570 and GDNF-luciferase plasmids; ** P < 0.01, vs . the GDNF-luciferase group (mean ± SD, n ≥ 3, independent sample t -test).

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: Derivative Assay, Activity Assay, Luciferase, Transfection, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation

doi: 10.3389/fcell.2023.1111705

Figure Lengend Snippet: Immunolabelling of PITX3, TUBB3/MAP2 in LMX1A-eGFP cultures. Panel (A) shows, from left to right, DAPI nuclear labelling (blue), LMX1A-eGFP fluorescence (green), PITX3 (red) and TUBB3/MAP2 in the same channel (white) and a color combined image (merged). Panels (B–D) show the corresponding data, but following cultivation in the absence of DAPT, dbcAMP and TGFβ3, respectively. Scale bar indicates 100 μm. Panel (E) shows LMX1A-eGFP fluorescence as a fraction of DAPI fluorescence ( n = 3). Panel (F) shows a typical Western blot while panel G shows changes in PITX3 expression following removal of additives ( n = 3). Bar graphs show mean +/- s. e. mean; * = p < 0.05.

Article Snippet: Cells were blocked with 3% (v/v) donkey serum in permeabilization solution 2 following incubation with primary antibody rabbit anti-PITX3 (1:100 Invitrogen), sheep anti-TH (1:500 Abcam), goat anti-GIRK2 (1:100 Abcam), rabbit anti-WNT5A (1:200 Abcam), mouse anti-TUBB3/chicken anti-MAP2 (1:1000 Abcam) in 3% donkey serum (1:100 dilution) overnight at 4°C.

Techniques: Fluorescence, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Generation of functional dopaminergic neurons from human spermatogonial stem cells to rescue parkinsonian phenotypes

doi: 10.1186/s13287-019-1294-x

Figure Lengend Snippet: Primer sequences used for qPCR

Article Snippet: The primary antibodies used in this study were as follows: UCHL1 (AbDSerotec), GFRA1, EN-1, OTX2 (Santa Cruz), GPR125, Tuj-1, PLZF, FoxA2 Lmx1a, Lmx1b (Abcam), DAT (Millipore), Curr1 (Millipore), PitX3 (GeneTex), and SYN (Chemicon).

Techniques: