Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 1. Nr2e3 was selected as a potential transcriptional factor of Tet2 expression. A–C) Mice were treated with different stressors daily to induce CMS mice. The FST (A), the TST (B) and the NSFT (C) were performed on control mice and CMS mice (n = 5). D) The hippocampal tissues were collected from control mice and CMS mice. The mRNA levels of 10 potential transcription factors of Tet2 were examined by qPCR analysis on control and CMS mice (n = 5). E) Tet2 and Nr2e3 protein levels were examined by Western blot. Representative blots of Tet2 and Nr2e3 protein after CMS were shown. F, G) Statistical analysis of F) Tet2 protein level and G) Nr2e3 level were shown (n = 5). All data were presented as mean ± SEM. A, B, C, F, G by using Student’s t-test. D)by using two-way ANOVA with Tukey’s multiple comparison tests (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Expressing, Control, Western Blot, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 2. Nr2e3 positively regulated Tet2 expression in HT-22 cells. A) After knockdown Nr2e3 in HT22 cells through transient infection of sgRNA and Cas9 expression lentivirus, Tet2 and Nr2e3 protein levels were examined by Western blot. Representative blots of Tet2 and Nr2e3 proteins were shown. B, C) Quantitative analysis of B) Nr2e3 protein level and C) Tet2 protein level were performed (n = 3). D) HT22 cells were infected with Nr2e3-3×Flag lentivirus. After puromycin screening and single clone isolation, the Nr2e3 protein level in Nr2e3OE cells was determined by Western blot with an anti- flag antibody. E) Nr2e3 and F) Tet2 mRNA levels in normal HT22 cells (Con) and Nr2e3OE cells were analyzed by qPCR (n = 3). G) The protein levels of Nr2e3 and Tet2 were examined by Western blot in normal HT22 cells (Con), HT22 Nr2e3KO cells, and HT22 Nr2e3KO cells infected with Nr2e3 lentivirus (rescue cells). H) Nr2e3 protein levels in different cells was analyzed (n = 4). I) Quantitative analysis of Tet2 protein level was performed (n = 4). J) qPCR analysis of Tet2 mRNA level in different cells were examined (n = 3). All data were presented as mean ± SEM. B, C, E, F) by using Student’s t-test. H, I, J) by using two-way ANOVA with Tukey’s multiple comparison tests (*p < 0.05, **p < 0.01 and ***p < 0.001).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Expressing, Knockdown, Infection, Western Blot, Isolation, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 3. Nr2e3 bound to the Tet2 promoter directly and enhanced the activity of the Tet2 promoter. A) Different doses (0.25, 0.5, 1.0, 1.5, and 2.0 μg) of Nr2e3 plasmid were co-transfected with full-length Tet2 promoter (Tet2 pro FL) in 293T cells. Dual-luciferase reporter assay was performed to measure the activity of the Tet2 promoter at 48 h after transfection (n = 3). B) Full-length Tet2 promoter plasmid was transfected into HT22 Nr2e3KO cell line. Dual-luciferase reporter assay was performed at 48 h after transfection (n = 3). C) The predicted Nr2e3 binding motifs (motif 10.3 and motif 11.6) at the Tet2 promoter by Jaspar (vs UCSD NCBI37 mm9) were shown. Different Tet2 promoter plasmids containing different truncated Tet2 promoters were constructed. D) Truncated Tet2 promoter plasmid and control plasmids were transfected into 293T cells. Dual-luciferase reporter assay was performed to measure the activity of Tet2 promoter at 48 h post-transfection (n = 3). E) Full-length Tet2 promoter plasmid (with motif-10.3) and Tet2 promoter plasmid without motif-10.3 (Tet2 pro-1608) were separately co-transfected into HEK293T cells with Nr2e3 plasmid. Nr2e3 monoclonal antibody and IgG antibody were used to perform the CHIP. The captured genomic DNA by Nr2e3 antibody was used to amplify PCR product containing Tet2 motif-10.3. F, G) The hippocampus of CMS mice and control mice were collected. Nr2e3 monoclonal antibody was used to perform the CHIP-qPCR in the hippocampal lysates. The enrichment fold changes of F) motif-10.3 and G) motif-11.6 were analyzed by qPCR (n = 3). All data were presented as mean ± SEM. A) by using one-way ANOVA with Tukey’s multiple comparison tests. B, D) by using two-way ANOVA with Tukey’s multiple comparison test. F, G by using Student’s t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Activity Assay, Plasmid Preparation, Transfection, Luciferase, Reporter Assay, Binding Assay, Construct, Control, ChIP-qPCR, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 4. Nr2e3 co-regulated the expression of Tet2 with DHX30 and LSD1. A, B) The hippocampal tissues of control mice and CMS mice were collected, then protein and mRNA were extracted. DHX30 mRNA level (A) and LSD1 mRNA level (B) were examined by qPCR analysis (n = 4). C) DHX30 plasmid was transfected in HT22 Nr2e3OE cells, and Tet2 mRNA level was examined by qPCR analysis at 48 h after transfection (n = 5–6). D) LSD1 plasmid was transfected in HT22 Nr2e3OE cells. Tet2 mRNA level was examined by qPCR analysis at 48 h after transfection (n = 4). E) DHX30 plasmid was transfected in HT22 Nr2e3KO cells, and then the Tet2 mRNA level was examined by qPCR analysis at 48 h after transfection (n = 6). F) LSD1 plasmid was transfected in HT22 Nr2e3KO cells. Tet2 mRNA level was examined by qPCR analysis at 48 h after transfection (n = 4). G) DHX30 plasmid was co-transfected with Nr2e3 in 293T cells. Tet2 promoter activity was detected using dual-luciferase reporter assay at 48 h after transfection (n = 3). H) LSD1 plasmid was co-transfected with Nr2e3 in 293T cells. Dual-luciferase reporter assay was performed to detect Tet2 promoter activity at 48 h after transfection (n = 3). I) DHX30 and LSD1 protein levels in the hippocampus were examined by Western blot following CMS. J, K) quantitative analysis of J) DHX30 and K) LSD1 protein levels was performed in control mice and CMS mice (n = 3). All data were presented as mean ± SEM. A–F, J, K) by using Student’s t-test. G, H) by using two-way ANOVA with Tukey’s multiple comparison tests (*p < 0.05 and ***p < 0.001).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Expressing, Control, Plasmid Preparation, Transfection, Activity Assay, Luciferase, Reporter Assay, Western Blot, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 5. The knockdown of Nr2e3 in the hippocampus resulted in depression-like behaviors in mice. A) A schematic diagram showed virus injection sites and the structure of the AAV plasmid. B) After 28 d of AAV-Nr2e3 RNAi virus injection into both sides of the hippocampus, protein levels of Nr2e3 and Tet2 were examined by Western blot. C, D) Quantitative analysis of C) Nr2e3 and D) Tet2 protein levels was performed (n = 7). E, F) At the same time, E) Nr2e3 and F) Tet2 mRNA levels were examined by qPCR at 28 d after viral injection (n = 5). G–I) The TST (G), the FST (H) and the NSFT (I) were performed from 21 d after viral injection (n = 15). L) The three-chamber social behavioral tests were also examined at 24 d after viral injection (n = 15). All data were presented as mean ± SEM. C–I) by using Student’s t-test. J by using two-way ANOVA with Tukey’s multiple comparison tests (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Knockdown, Virus, Injection, Plasmid Preparation, Western Blot, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 7. Nr2e3-Tet2 signal pathway was involved in hippocampal neuronal plasticity. A) The hippocampal tissues from Nr2e3 KD mice and control (NC) mice were collected for Western blot. Protein levels of PSD95, Syn1, and NMDAR1 in the hippocampus were detected. B–D) Protein levels of PSD95 (B, n = 7), NMDAR1 (C, n = 6), and Syn1 (D, n = 7) in the hippocampus were quantitatively analyzed. E) The spines of hippocampus neurons in control mice and Nr2e3 KD mice by Golgi staining were shown (scale bar, 20um). F) The quantitative analysis of spine number in the hippocampal neurons was performed (n = 60). G) Primary neurons were isolated and cultured for 4 d, and then the neurons were infected with AAV-Nr2e3 RNAi and control AAV-RNAi for 12 h. After an additional 8 d of culture, the neurons were harvested. The protein levels of Nr2e3, Tet2, PSD95, NMDAR1, and Syn1 were detected by Western blot. H–L) Quantitative analysis of protein levels of H) Nr2e3, I) Tet2, J) PSD95, K) NMDAR1, L) and Syn1 was performed (n = 3). All data were presented as mean ± SEM. Statistical significance was determined by using Student’s t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Control, Western Blot, Staining, Isolation, Cell Culture, Infection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 8. AZA increased Nr2e3 expression in HT22 cells and mice. A, B) HT22 cells were treated with AZA (10 μM) or DMSO. At different time points after AZA treatment (12, 24, and 48 h), cells were harvested, and A) Nr2e3 and B) Tet2 mRNA levels were examined by qPCR analysis. C. HT22 cells were treated with AZA (10 μM) or DMSO for 24 h. Protein levels for Tet2 and Nr2e3 were examined by Western blot. D, E) Quantitative analysis of protein levels for D) Nr2e3 and E) Tet2 was performed (n = 3). F) HT22 Nr2e3KO cells were treated with AZA (10 μm) or DMSO for 24 h. The mRNA level of Tet2 was examined by qPCR analysis. G, H) Mice were treated with AZA (5 mg kg−1 i.p) or same volume of vehicle. At different time points (1 h, 2 h, 4 h and 8 h) after AZA administration, the hippocampal tissues were collected. G) Tet2 and H) Nr2e3 mRNA levels was examined by qqPCR analysis (n =

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Expressing, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

doi: 10.1002/advs.202400726

Figure Lengend Snippet: Figure 9. AZA improved depression-like behaviors via the Nr2e3-Tet2 signaling pathway. A, B) Control mice and CMS mice were treated with AZA (5 mg kg−1 i.p) or the same volume of vehicle for 4 w. TST (A) and FST (B) were performed weekly (n = 10). C) After AZA administration for 28 d-, the hippocampal tissues were collected. Protein levels of PSD95 were determined by Western blot. D) Protein levels of PSD95 were quantified (n = 7). E) TST and F) FST were performed in control mice and Tet2 cKO mice (n = 9,15). G, H) Tet2 cKO mice were treated with AZA (5 mg kg−1 i.p) or the same volume of vehicle. G) TST and H) FST were performed weekly (n = 7–8). All data were presented as mean ± SEM. A, B, D, G, H) by using two-way ANOVA with Tukey’s multiple comparison tests. E, F) by using Student’s t-test (*p < 0.05, **p < 0.01 and ***p < 0.001 vs Con+vehicle group or WT group; #p < 0.05 and ###p < 0.001 vs CMS+ vehicle group).

Article Snippet: Plasmids Constructions: The full-length of mouse Tet2 promoter luciferase reporter plasmid (pGL3-Tet2 promoter F) was purchased from Addgene (63879, Addgene, MA, USA).

Techniques: Control, Western Blot, Comparison

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a – c Scratch assay in MDA-MB-231 and SUM159 cells treated with DMSO or KH-3. a , b Representative images of cell migration at 0 and 24 h after scratching with indicated treatment in MDA-MB-231 ( a ) and SUM159 ( b ) cells, scale bars: 50 μm. c Wound widths in two cell lines 24 h after scratching and treatment (*** P < 0.001, t -test, n = 3). d – f Invasion assay in MDA-MB-231 and SUM159 cells treated by DMSO, KH-3B or KH-3. d , e Representative images of stained invaded cells with indicated treatment in MDA-MB-231 ( d ) and SUM159 ( e ) cells, scale bars: 200 μm. f Invaded cell numbers per image in both cell lines with indicated treatment (*** P < 0.001, one-way ANOVA, n = 6). g The heatmap view of PCR pathway array focusing on invasion and metastasis related genes. The relative mRNA levels were presented as z score, each treatment was triplicated. h CDH1 luciferase reporter assay in HEK 293FT cells treated by DMSO, KH-3B or KH-3. Values are mean ± SD from n = 4 independent experiments (** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: For CDH1 reporter assay, HEK 293FT cells co-transfected with pGL3 vector with or without CDH1 promoter (Addgene, #61798) and renilla were treated with DMSO, KH-3 or KH-3B for 48 h. For FOXQ1 3′-UTR study, MDA-MB-231 and SUM159 cells transfected with pEZX-MT06 reporter vector with or without human FOXQ1 3′-UTR were treated with DMSO, KH-3 or KH-3B at indicated doses for 24 h. The cells were then harvested and assayed using the Dual-Glo Luciferase Assay (Promega).

Techniques: Wound Healing Assay, Migration, Invasion Assay, Staining, Luciferase, Reporter Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a Venn diagram depicting the number of targets identified in two independent RNA-seq experiments. FOXQ1 is a direct HuR target, which is also one of the top mRNAs decreased by KH-3 treatment. b Protein expression levels of HuR, FOXQ1 and E-cadherin in HMEC and a panel of TNBC cell lines. c , d Pull-down analysis of KH-3 disrupting ARE FOXQ1 oligo binding to endogenous HuR in MDA-MB-231 and SUM159 cells. c Representative western blot result from one experiment. d Quantified relative HuR expression. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, *** P < 0.001, one-way ANOVA). e , f RNP IP analysis of HuR bound FOXQ1 mRNA affected by KH-3 in MDA-MB-231 ( e ) and SUM159 ( f ) cells. Values are mean ± SD from three independent experiments (** P < 0.01, *** P < 0.001, one-way ANOVA). g , h Relative FOXQ1 mRNA levels in MDA-MB-231 ( g ) and SUM159 ( h ) cells treated with DMSO, KH-3 or KH-3B at the indicated time points. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA). i , j FOXQ1 3′-UTR luciferase reporter assay in MDA-MB-231 ( i ) and SUM159 ( j ) cells treated by DMSO, KH-3B or KH-3. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA).

Article Snippet: For CDH1 reporter assay, HEK 293FT cells co-transfected with pGL3 vector with or without CDH1 promoter (Addgene, #61798) and renilla were treated with DMSO, KH-3 or KH-3B for 48 h. For FOXQ1 3′-UTR study, MDA-MB-231 and SUM159 cells transfected with pEZX-MT06 reporter vector with or without human FOXQ1 3′-UTR were treated with DMSO, KH-3 or KH-3B at indicated doses for 24 h. The cells were then harvested and assayed using the Dual-Glo Luciferase Assay (Promega).

Techniques: RNA Sequencing, Expressing, Binding Assay, Western Blot, Luciferase, Reporter Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a , b Invasion assay in parental MDA-MB-231 cells, sgControl and two HuR KO clones transfected with control vector or vector containing FOXQ1 cDNA. a Representative images of stained invaded cells, scale bars: 200 μm. b The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). c mRNA expression levels of FOXQ1, CDH1 and CD82 in parental MDA-MB-231 cells, sgControl and two HuR KO clones transfected with control vector or vector containing FOXQ1 cDNA. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, *** P < 0.001, two-way ANOVA). d , e Invasion assay in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with DMSO or 10 μM KH-3 treatment. d Representative images of stained invaded cells, scale bars: 200 μm. e The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). f mRNA expression levels of FOXQ1, CDH1 and CD82 in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with treatment of DMSO or 10 μM KH-3. Values are mean ± SD from n = 3 independent experiments (** P < 0.01, *** P < 0.001, two-way ANOVA). g , h Protein expression levels of FOXQ1, Bcl-2, Msi2, β-catenin, and HuR in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with treatment of DMSO or KH-3 at the indicated doses for 48 h. α-Tubulin is used as loading control. g Representative western blot results from one experiment. h Quantified relative expression of HuR and downstream targets. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: For CDH1 reporter assay, HEK 293FT cells co-transfected with pGL3 vector with or without CDH1 promoter (Addgene, #61798) and renilla were treated with DMSO, KH-3 or KH-3B for 48 h. For FOXQ1 3′-UTR study, MDA-MB-231 and SUM159 cells transfected with pEZX-MT06 reporter vector with or without human FOXQ1 3′-UTR were treated with DMSO, KH-3 or KH-3B at indicated doses for 24 h. The cells were then harvested and assayed using the Dual-Glo Luciferase Assay (Promega).

Techniques: Invasion Assay, Clone Assay, Transfection, Control, Plasmid Preparation, Staining, Expressing, Western Blot