Journal: iScience

Article Title: Otic organoids: A model to study spiral ganglion neuron characteristics in Tmprss3-deficiency

doi: 10.1016/j.isci.2025.114355

Figure Lengend Snippet: Stepwise differentiation of iPSCs into SGN-enriched organoids and early phenotypic differences in TMPRSS3-deficient clones (A) Schematic overview of the stepwise differentiation protocol. Human iPSCs were expanded in E8 medium, transferred to serum-free (SF) medium, and subsequently directed toward the otic lineage using DFNB medium under feeder-free conditions. (B) Phase-contrast images showing morphological changes during differentiation: adherent iPSC colonies (iPSCs), early aggregates (day 6 and day 9), spherical organoids (day 15), and progressively larger structures (day 25, day 45, and day 70). (C–N) Pluripotency validation of K2/8 WT (C–H) and K2/8 C4 KO (I–N) iPSCs. Nuclear markers NANOG (C, I), OCT4 (D, J), SOX2 (E, K) and surface markers SSEA4 (F, L), TRA-1-60 (G, M), TRA-1-81 (H, N) confirm robust pluripotency of all lines. (O–V) Early otic differentiation at days 3, 6, 9, and 11 in K2/8 WT (O, Q, S, U) and K2/8 C4 KO (P, R, T, V) organoids. (O, P) Day 3: K2/8 WT organoids show diffuse EYA1, ECAD + epithelial boundaries, and DLX5 + cells, whereas K2/8 C4 organoids display perinuclear EYA1, larger ECAD + cells, and no DLX5. (Q, R) Day 6: K2/8 WT retains strong nuclear SOX2 and ECAD; K2/8 C4 shows loss of SOX2 + progenitors and collapsed ECAD expression. (S, T) Day 9: K2/8 WT exhibits nuclear PAX8 + progenitors within organized ECAD + epithelium; K2/8 C4 shows condensed ECAD and reduced PAX8.U, V) Day 11: K2/8 WT expresses not PAX8 and JAG1; K2/8 C4 shows early JAG1 and absent PAX8 expression. Representative images were obtained from n ≥ 3 organoids per time point from four independent differentiation experiments. Scale bars, 20 μm (B: iPSCs, d6, d9, C-N, O′-V′), 100 μm (B: d15, d25, O-V), and 500 μm (B: d45, d70). Nuclei were counterstained with DAPI (blue). See also .

Article Snippet: EYA1 , ProteinTech , 22658-1-AP.

Techniques: Clone Assay, Biomarker Discovery, Expressing

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1 potentially dephosphorylates BCL2L12 at T33 in glioma cells. A, Scheme flow chart of phosphoproteomic analysis. B, Criteria for searching novel substrate of EYA1 in glioma cells. C, Table showing the changes of phosphorylation level of representative peptides in response to EYA1 overexpression. Phosphorylation sites were highlighted with red color. “TPXXSP” motif is highlighted with black underline. D, Sequence alignment of potential EYA1-regulated region in BCL2L12 among various species. E, T33 was a potential dephosphorylation site of BCL2L12 by EYA1. HEK293T cells were transfected with wild-type or various phospho-dead mimic mutations of BCL2L12. Cells were lysed and subjected to IP using anti-flag beads and immunoblotting using p-TP specific antibody. F, Schematic diagram of BCL2L12 “T-P” dipeptide and phosphor-dead mimic mutants. G-I, Quantitative statistic results of the phosphorylation level of wild-type and phospho-dead mimic mutations of BCL2L12 with or without EYA1 overexpression. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Phospho-proteomics, Over Expression, Sequencing, De-Phosphorylation Assay, Transfection, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1 interacts and colocalizes with BCL2L12 in glioma cells. A, Co-IP showing physical interaction between Flag-EYA1 and HA-BCL2L12 in HEK293T cells. B, Co-IP showing physical interaction between Flag-EYA1 and HA-BCL2L12 in U87MG cells. C, Live-cell imaging shows that EYA1 (green) colocalizes with BCL2L12 (red) in HEK293T cells. Bar=20 μm. D, Live-cell imaging shows that EYA1 (green) colocalizes with BCL2L12 (red) in U87MG cells. Bar= 20 μm. E, Schematic diagram of EYA1 truncations. F, Mapping essential domains mediating protein-protein interaction between EYA1 and BCL2L12.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Co-Immunoprecipitation Assay, Live Cell Imaging

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1 regulates BCL2L12 turnover. A, Immunoblotting showing the level of BCL2L12 protein is elevated with EYA1 amount increasing. B-D, HEK293T cells were transfected with HA-EYA1 and Flag-tagged wild-type (B) or T33A (C) BCL2L12. 24 hours later, cells were treated with protein synthesis inhibitor cycloheximide as indicated. Protein level was detected, and protein stability curve was showed (D). E, Immunoblotting showing the level of BCL2L12 protein is restored by MG132 treatment in T98G and U87MG cells. F, BCL2L12 was modified by ubiquitination. HEK293T cells were transfected with HA-ubiquitin and BCL2L12 under MG132 treatment. Cells were lysed and subjected to IP using anti-flag beads and immunoblotting. G, IP-immunoblotting showing BCL2L12 was modulated by K48-polyubiquitination under MG132 treatment. H, EYA1 suppressed BCL2L12 ubiquitination. HEK293T cells were transfected with Flag-BCL2L12, HA-EYA1, His-Ub and treated with MG132 as indicated. Cells were lysed and subjected to Flag IP and immunoblotting using indicated antibodies. I, Ubiquitination assay showing the ubiquitination level of wild-type, phosphor-mimic (T33E and T33D), and phosphor-dead (T33A) BCL2L12 under MG132 treatment.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Western Blot, Transfection, Modification, Ubiquitin Proteomics

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1 is essential for glioma cell growth in vitro . A-B, T98G and U87MG cells transfected with siControl or siEYA1 (-1 and -2) were subjected to cell proliferation assay. Corresponding quantitative results are shown (B). C-D, T98G and U87MG cells transfected with siControl or siEYA1 (-1 and -2) were subjected to clone formation assay. Corresponding quantitative results are shown (D). E-F, U87MG and U251MG cells transfected with siControl or siEYA1 (-1 and -2) were subjected to tumorsphere formation assay. Corresponding quantitative results are shown (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: In Vitro, Transfection, Proliferation Assay, Tube Formation Assay

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1 is essential for glioma tumor formation in vivo . A-C, GL261 cells stably expressing shControl or shEya1 were subcutaneously injected into the right flanks of nude mice as indicated (n = 5 each group). After 32 days of injection, mice were sacrificed, and xenograft tumors were removed. Representative tumor images (A), tumor volume development curve (B), and tumor weight (C) are shown. D-G, GL261 cells stably expressing shControl or shEya1 were orthotopically transplanted into mice brain as indicated (n = 8 each group). Representative luminescence images (D), H.E. staining of mice brain tumor (E), survival curve (F), and statistical analysis of tumor luminescence (G) are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: In Vivo, Stable Transfection, Expressing, Injection, Staining

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1-BCL2L12 signaling pathway is essential for glioma development. A, T98G and U87MG cells transfected with siControl or siEYA1 were subjected to immunoblot analysis and BCL2L12 antibody was used to detect the endogenous BCL2L12 protein level. B, U87MG and U251MG cells overexpressing EYA1 were subjected to immunoblot analysis and BCL2L12 antibody was used to detect the endogenous BCL2L12 protein level. C, BCL2L12 is overexpressed in the EYA1-silencing cells in T98G and U87MG and cell lysates were subjected to immunoblot analysis for validation. D-G, T98G and U87MG cells transfected with indicated siRNAs or plasmids were subjected to CCK-8 assay (D and E). Corresponding quantitative results (F and G) are shown. H-J, T98G and U251MG cells transfected with indicated siRNAs or plasmids were subjected to clone formation assay (H). Corresponding quantitative results (I and J) are shown. K-M, U251MG cells stably expressing empty vector, wild-type, phosphor-mimic (T33E), and phosphor-dead (T33A) BCL2L12 were subcutaneously injected into the right flanks of nude mice as indicated (n = 5 each group). After 32 days of injection, mice were sacrificed, and xenograft tumors were removed. Representative tumor images (K), tumor volume development curve (L), and tumor weight (M) are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Transfection, Western Blot, Biomarker Discovery, CCK-8 Assay, Tube Formation Assay, Stable Transfection, Expressing, Plasmid Preparation, Injection

Journal: International Journal of Biological Sciences

Article Title: Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development

doi: 10.7150/ijbs.99619

Figure Lengend Snippet: EYA1 and BCL2L12 positively correlate in glioma patient samples. A, Representative immunohistochemical staining of EYA1 and BCL2L12 proteins in glioma patient samples from low to high level. B, Pearson correlation analysis of EYA1 and BCL2L12 protein expression in glioma patient samples. C-D, Kaplan-Meier analysis of the overall survival (C) and progression-free survival (D) of glioma patients with high and low BCL2L12 expression from TCGA cohort. E, The proposed working model. This image was created with BioRender.com.

Article Snippet: The antibody against EYA1 (Proteintech, 22658-1-AP) and BCL2L12 (Proteintech, 16612-1-AP) were utilized for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Nature communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: Fig. 7 | Model depicting dephosphorylation mediated regulation of PLK1 by EYA4/EYA1. G2, Phosphorylation of the putative PDS on EYA4/EYA1 (S128 on EYA4) allows for interaction with PLK1 and dephosphorylation of pY445 within the PBD. G2→Prophase, Dephosphorylation of pY445 changes the structure of the PLK1 PBD, favouring the interaction between PLK1 and PLK1 activation complex members, and permitting greater levels of pT210 phosphorylation by AURKA. Prophase, Active PLK1 supports centrosome maturation and separation in preparation for mitotic spindle formation. Prophase→Successful Mitosis, Accurate and timely completion of mitosis. Alternatively, EYA4/EYA1 loss or EYA inhibition causes mitotic aberra- tions, increased mitotic duration, and mitotic cell death. Created with biorender.com.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192A302324ABethyl (1:1000),Aurka 12100Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Activation Assay, Inhibition