Trim69 was expressed in zebrafish embryos’ brain.

(a-d) trim69 was expressed in zebrafish embryo brain at 24hpf; (e-h) trim69 was expressed in zebrafish embryo brain at 48hpf; (i-l) trim69 was expressed in zebrafish embryo brain at 72hpf; e: eye; fb: forebrain; mb: midbrain; hb: hindbrain; finb: fin bud; ba: brachial arch; scale bar: 200 µm.

Trim69 knockdown induces deformed brain and apoptosis.

(A) The zebrafish embryo phenotype after trim69 knock-down. trim69-CON(a): control group; trim69-MO(b): trim69 knocking down; trim69-MO:trim69-mRNA(c): co-injection with trim69-MO and human trim69 mRNA; black arrow indicates mid-hind brain boundary(MHB); scale bar: 200 µm; (d) Statistical analysis of the zebrafish embryo phenotype. Normal: the phenotype of the zebrafish embryo is normal; deformed: the phenotype of the zebrafish embryo phenotype is abnormal. (B) The examined apoptosis of zebrafish embryos by TUNEL assay. trim69-CON(e): control group; trim69-MO(f): trim69 knocking down; trim69-MO:trim69-mRNA(g): co-injection with trim69-MO and human trim69 mRNA; black arrow indicates apoptotic cells; scale bar: 200 µm.

Trim69 knockdown affects zebrafish embryo neurogenesis.

(A) The expression of neuronal differentiation markers was decreased in response to trim69 knockdown. trim69-CON: control group; trim69-MO: trim69 knocking down; black arrow indicates positive signals; scale bar: 200 µm; (B) The expression of stem cell markers was decreased in response to trim69 knockdown. trim69-CON: control group; trim69-MO: trim69 knocking down; black arrow indicates positive signals; scale bar: 200 µm.

TRIM69 negatively regulates the expression of c-Jun.

(A) HEK 293T cells were transfected with Flag-TRIM69 or a control expression vector. The cell lysates were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (B) HEK 293T cells were transfected with Flag-TRIM69 or a control expression vector. At 36 h post-transfection, the cells were treated with cycloheximide (200 µg/ml, CHX) and MG132 (20 µM) or chloroquine (100 µM) for 2 h. (C) HEK 293T cells were co-transfected with Flag-TRIM69 along with siRNA-NC or siRNA-TRIM69. The cell lysates were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (D,E) SH-SY5Y and HEK 293T cells were transfected with siRNA-NC or siRNA-TRIM69. The cell lysates were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. Simultaneously, the relative RNA level of TRIM69 was determined by qPCR. Data were measured in triplicate and statistically analyzed by unpaired t-test, p < 0.01; values and bars represent the mean and standard deviation, respectively; (F,G) trim69 knockdown decreased the expression of c-Jun in zebrafish. trim69-CON: control group; trim69-MO: trim69 knocking down; scale bar: 200 µm.

Loss of c-Jun rescued the deformed brain and apoptosis induced by trim69 knockdown.

(A) Loss of c-Jun rescued the deformed brain induced by trim69 knockdown. trim69-CON(a): control group; trim69-MO(b): trim69 knocking down; trim69-MO:c-Jun-MO(c): co-injection with trim69-MO and c-Jun-MO; black arrow indicates mid-hind brain boundary(MHB); scale bar: 200 µm; (d) Statistical analysis of the zebrafish embryo phenotype. Normal: the phenotype of the zebrafish embryo is normal; deformed: the phenotype of the zebrafish embryo phenotype is abnormal. (B) The examined apoptosis of zebrafish embryos by TUNEL assay. trim69-CON(e): control group; trim69-MO(f): trim69 knocking down; trim69-MO:cJun-MO(g): co-injection with trim69-MO and c-Jun-MO; black arrow indicates apoptotic cells; scale bar: 200 µm.

Loss of c-Jun rescued the zebrafish embryo neurogenesis.

(A) Loss of c-Jun rescued the expression of neuronal differentiation markers. trim69-MO(f): trim69 knocking down; trim69-MO:cJun-MO(g): co-injection with trim69-MO and c-Jun-MO; black arrow indicates positive signals; scale bar: 200 µm; (B) Loss of c-Jun rescued the expression of stem cell markers. trim69-MO(f): trim69 knocking down; trim69-MO:cJun-MO(g): co-injection with trim69-MO and c-Jun-MO; black arrow indicates positive signals; scale bar: 200 µm.

Trim69 knockdown induces deformed brain. trim69-CON: control group; trim69-MO: trim69 knocking down; trim69-MO:trim69-mRNA: co-injection with trim69-MO and human trim69 mRNA;trim69-MO:c-Jun-MO: co-injection with trim69-MO and c-Jun-MO; black arrow indicates mid-hind brain boundary(MHB); scale bar: 200µm.

Expression of brain region specific markers detected byin situhybridization. Detection of the expression ofotx2 (marker of forebrain and MHB), eng2a (marker for MHB and hindbrain), egr2b (marker for MHB and hindbrain), and fgf8a (marker for forebrain and MHB) after loss oftrim69 by in situ hybridization.trim69-CON: control group; trim69-MO: trim69 knocking down; black arrow indicates positive signals; scale bar: 200µm.