Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 1. TWAS and regional association plots for kidney function and DACH1 expression. (A) Conceptual model for TWAS. (B–D) Regional association plots for DACH1 locus (index SNP; rs626277). (B) eGFR GWAS data from CKDGen study. (C) eQTL data in kidney tubules. (D) eQTL data in kidney glomeruli. Each dot represents 1 SNP. The dots are colored according to their relationship to the index SNP (rs626277). The red dots indicate high correlation (r2 > 0.8) (LD) with the index SNP. The left y axis indicates ––log10 (P value). The right y axis indicates recombination rate (cM/Mb). The red dotted hori- zontal line in the GWAS regional plot indicates the genome-wide significance threshold (5 × 10−8). Local permutation-based P value was used to define significance for the eQTL (tubule; P = 8.5 × 10−6, eQTL glom; P = 1.2 × 10−5). The x axis indicates the genomic location on chromosome 13. The arrow indicates the transcriptional direction for DACH1. (E) Box plot: the x axis represents the SNP (rs626277 genotype A/A, A/C, C/C), and the y axis shows relative DACH1 expression (P = 5.61 × 10–08) in human kidney tubule samples. (F) Effect sizes of eGFR GWAS (CKDGen) SNPs (y axis) were plotted against eQTL SNPs (x axis). Error bars show the standard errors of SNP effects. Significant SNPs in SMR analysis were plotted. Red triangle indicates the top eQTL signal. (G) Venn diagram of TWAS, SMR, and MetaXcan listing the 10 genes prioritized by all 3 methods.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Genome Wide

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 3. DACH1 expression in mouse and human kidneys. (A–D) Represen- tative immunofluorescence staining of DACH1 (red), DAPI (blue), and tubule- specific markers (green) in healthy adult mouse kidneys. Markers for proximal tubule, LTL (A) and AQP1 (B); distal tubule, Ksp-cadherin (KSP) (C); and col- lecting tubule, AQP2 (D). The right panels in A–D show higher magnification images of the regions within the white-dotted frames. G, glomerulus. (E–H) Represen- tative immunofluorescence staining for DACH1 (red), DAPI (blue), and tubule-spe- cific markers (green) in healthy human kidney. Markers for proximal tubule, LTL (E); thick ascending loop of Henle, UMOD (F); distal tubule, CAL (G); and collecting tubule, AQP2 (H). Right panels in E–H show higher magnification images of the regions within the white-dotted frames. Scale bars: 20 μm.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 6. Mice with tubule-specific transgenic expression of Dach1 is protected from fibrosis. (A) Experimental design: Pax8rtTA/TRE-Dach1 (Dach1 OE) mice and control littermates (WT) were placed on doxycycline-containing chow. Mice were injected with FA or NaHCO3 (sham) i.p. Kidneys and serum were collected 7 days after injection. (B) Relative Dach1 mRNA expression in whole kidney tissue samples. (C–E) Profibrotic gene expression in whole kidney tissue samples: Col1a1 (C), Col3a1 (D), Fn1 (E). Gene expression levels were normalized to Gapdh. (F) Representative images of Sirius red staining. Scale bar: 20 μm. (G) Quantitative analysis (as a percentage of positive area) of Sirius red staining of kidney sections. (H) Representative images of PAS-stained kidney sections. Scale bar: 20 μm. (I) BUN levels. Sham-treated group: WT (n = 6), Dach1 OE (n = 6); FA-treated group: WT (n = 6), Dach1 OE (n = 8). Light gray bars represent WT; red bars represent Dach1 OE. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Transgenic Assay, Expressing, Control, Injection, Gene Expression, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 8. DACH1 transcriptionally controls cell cycle genes in kidney tubule cells. (A) DACH1-ChIP 19,460-binding sites, corresponding to 5111 genes in the K562 cell (human immortalized myelogenous leukemia). Lower panel shows the distribution of peaks compared with TSS distance. (B) Top 10 most significant pathways from the Molecular Signatures Database (MsigDB) using the Genomic Region of Enrichment of Annotations (GREAT) package (see Supplemental Methods). (C) Primary mouse kidney tubule cells were isolated from Dach1fl/fl mice and infected with adenovirus-GFP (Ade-GFP; control [CTRL]) or adenovirus-Cre-GFP (Ade-Cre-GFP; Dach1 KO). Representative Western blots of DACH1 protein expression in control and Dach1 KO tubule cells. GAPDH was used as loading control. (D) DACH1-ChIP-PCR for Ccnd1 and Ccnd2 in control kidney tubule cells. Three independent experiments were performed. (E) Integrated Genome Browser view of the human CCND2 locus. From top to bottom, the labels indicate the CCND2 locus, DACH1–ChIP-Seq, followed by human kidney snATAC-Seq in DCT, proximal tubule, loop of Henle, principal cell, intercalated cell. The box highlights the overlapping peaks between DACH1–ChIP-Seq and snATAC-Seq. (F) Relative gene expression of Ccna1, Ccnd1, Ccnd2, and Ccne1 in Ade-GFP or Ade-Cre-GFP transfected cells (n = 3). Gapdh was used as internal control. (G) Results of cell proliferation analysis using the MTT assay at 24 and 48 hours after Ade-GFP or Ade-Cre-GFP infection. (n = 3 in triplicate). *P < 0.05, **P < 0.01, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Binding Assay, Isolation, Infection, Control, Western Blot, Expressing, ChIP-sequencing, Gene Expression, Transfection, MTT Assay

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 9. DACH1 controls cell cycle in vivo. (A and B) Relative transcript expression of Ccnd2 (A) and Mki67 (B) in whole kidney samples of sham-treated and FA-injected WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/ Dach1fl/fl (Dach1 KO) mice. (C and D) Representative Ki-67 immunostaining (C) and quantification of Ki-67–positive cells per high-power field in kidney tissue of sham-treated and FA-inject- ed WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/Dach1fl/fl (Dach1 KO) mice. Scale bar: 20 μm. (E and F) Relative transcript levels of Ccnd2 (E) and Mki67 (F) in whole kidney samples of sham-treated and FA-injected WT and Pax8rtTA/TRE-Dach1 (Dach1 OE) mice. (A–D) Sham-treated group: WT (n = 4), Dach1 HZ (n = 4), Dach1 KO (n = 4); FA-treated group: WT (n = 10), Dach1 HZ (n = 8), Dach1 KO (n = 8). Light gray bars represent WT, red bars represent Dach1 HZ, and green bars represent Dach1 KO group. (E and F) Sham-treat- ed group: WT (n = 6), Dach1 OE (n = 6); FA-treated group: WT (n = 6), Dach1 OE (n = 8). Light gray bars represent WT,; red bars represent Dach1 OE. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: In Vivo, Expressing, Injection, Immunostaining

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 10. DACH1 transcriptionally controls cytokine expression in cultured kidney tubule cells. (A–C) DACH1-ChIP-qPCR for Ccl2 (A), Csf1 (B), Icam1 (C) using primary mouse kidney tubule epithelial cells. The y axis is presented as percentage of input (n = 3). (D) Integrated Genome Browser view of the human CSF1 locus. From top to bottom, the labels indicate CSF1 locus, DACH1-ChIP-seq, followed by human kidney snATAC-Seq (open chromatin region) in DCT, proximal tubule, loop of Henle, principal cells, intercalated cells. The red box highlights the overlapping peaks of DACH1 ChIP-Seq and human kidney snATAC-Seq. (E) The relative expression of Cxcl1, Cxcl2, Ccl2, Ccl5, Tnfa, Csf1, Il1b, Il17c, Il27, and Icam1 in Ade-GFP or Ade-Cre-GFP transfected renal tubule cells from Dach1fl/fl mice (n = 3). Gapdh was used as internal control. (F) Experimental scheme of Raw 264.7 macrophage chemotaxis assay. The supernatants of Ade-GFP or Ade- Cre-GFP-transfected renal tubule cells from Dach1fl/fl mice were added to the lower chamber. The number of macrophages (macrophage migration) was ana- lyzed by relative fluorescence units (RFU) after 2 or 24 hours (n = 3 in triplicate). *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Cell Culture, ChIP-qPCR, ChIP-sequencing, Transfection, Control, Chemotaxis Assay, Migration, Fluorescence

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 11. DACH1 controls cytokine expression and macrophage infiltration. (A–C) Relative transcript expression of Ccl2 (A), Csf1 (B), and Icam1 (C) in kidneys of sham-treated and FA-treated WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/Dach1fl/fl (Dach1 KO) mice. (D–E) Representative immunostaining with F4/80 (macrophage marker) (D) and quantification of F4/80-positive area (E) in kidney tissue of sham-treated and FA-treated WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/Dach1fl/fl (Dach1 KO) mice. Scale bar: 20 μm. (F–H) Relative transcript expression of Ccl2 (F), Csf1 (G), and Icam1 (H) in kidneys of sham-treated and FA-treated WT and Pax8rtTA/TRE-Dach1 (Dach1 OE) mice. Gapdh was used as internal control. (A–D) Sham-treated group: WT (n = 10), Dach1 HZ (n = 10), Dach1 KO (n = 10); FA-treated group: WT (n = 10), Dach1 HZ (n = 8), Dach1 KO (n = 8). Light gray bars represent WT, red bars represent Dach1 HZ, and green bars represent Dach1 KO. (E and F) Sham-treated group: WT (n = 6), Dach1 OE (n = 6); FA-treated group; WT (n = 6), Dach1 OE (n = 8). Light gray bars represent WT; red bars represent Dach1 OE. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Immunostaining, Marker, Control

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 1. TWAS and regional association plots for kidney function and DACH1 expression. (A) Conceptual model for TWAS. (B–D) Regional association plots for DACH1 locus (index SNP; rs626277). (B) eGFR GWAS data from CKDGen study. (C) eQTL data in kidney tubules. (D) eQTL data in kidney glomeruli. Each dot represents 1 SNP. The dots are colored according to their relationship to the index SNP (rs626277). The red dots indicate high correlation (r2 > 0.8) (LD) with the index SNP. The left y axis indicates ––log10 (P value). The right y axis indicates recombination rate (cM/Mb). The red dotted hori- zontal line in the GWAS regional plot indicates the genome-wide significance threshold (5 × 10−8). Local permutation-based P value was used to define significance for the eQTL (tubule; P = 8.5 × 10−6, eQTL glom; P = 1.2 × 10−5). The x axis indicates the genomic location on chromosome 13. The arrow indicates the transcriptional direction for DACH1. (E) Box plot: the x axis represents the SNP (rs626277 genotype A/A, A/C, C/C), and the y axis shows relative DACH1 expression (P = 5.61 × 10–08) in human kidney tubule samples. (F) Effect sizes of eGFR GWAS (CKDGen) SNPs (y axis) were plotted against eQTL SNPs (x axis). Error bars show the standard errors of SNP effects. Significant SNPs in SMR analysis were plotted. Red triangle indicates the top eQTL signal. (G) Venn diagram of TWAS, SMR, and MetaXcan listing the 10 genes prioritized by all 3 methods.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Genome Wide

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 3. DACH1 expression in mouse and human kidneys. (A–D) Represen- tative immunofluorescence staining of DACH1 (red), DAPI (blue), and tubule- specific markers (green) in healthy adult mouse kidneys. Markers for proximal tubule, LTL (A) and AQP1 (B); distal tubule, Ksp-cadherin (KSP) (C); and col- lecting tubule, AQP2 (D). The right panels in A–D show higher magnification images of the regions within the white-dotted frames. G, glomerulus. (E–H) Represen- tative immunofluorescence staining for DACH1 (red), DAPI (blue), and tubule-spe- cific markers (green) in healthy human kidney. Markers for proximal tubule, LTL (E); thick ascending loop of Henle, UMOD (F); distal tubule, CAL (G); and collecting tubule, AQP2 (H). Right panels in E–H show higher magnification images of the regions within the white-dotted frames. Scale bars: 20 μm.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 6. Mice with tubule-specific transgenic expression of Dach1 is protected from fibrosis. (A) Experimental design: Pax8rtTA/TRE-Dach1 (Dach1 OE) mice and control littermates (WT) were placed on doxycycline-containing chow. Mice were injected with FA or NaHCO3 (sham) i.p. Kidneys and serum were collected 7 days after injection. (B) Relative Dach1 mRNA expression in whole kidney tissue samples. (C–E) Profibrotic gene expression in whole kidney tissue samples: Col1a1 (C), Col3a1 (D), Fn1 (E). Gene expression levels were normalized to Gapdh. (F) Representative images of Sirius red staining. Scale bar: 20 μm. (G) Quantitative analysis (as a percentage of positive area) of Sirius red staining of kidney sections. (H) Representative images of PAS-stained kidney sections. Scale bar: 20 μm. (I) BUN levels. Sham-treated group: WT (n = 6), Dach1 OE (n = 6); FA-treated group: WT (n = 6), Dach1 OE (n = 8). Light gray bars represent WT; red bars represent Dach1 OE. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Transgenic Assay, Expressing, Control, Injection, Gene Expression, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 8. DACH1 transcriptionally controls cell cycle genes in kidney tubule cells. (A) DACH1-ChIP 19,460-binding sites, corresponding to 5111 genes in the K562 cell (human immortalized myelogenous leukemia). Lower panel shows the distribution of peaks compared with TSS distance. (B) Top 10 most significant pathways from the Molecular Signatures Database (MsigDB) using the Genomic Region of Enrichment of Annotations (GREAT) package (see Supplemental Methods). (C) Primary mouse kidney tubule cells were isolated from Dach1fl/fl mice and infected with adenovirus-GFP (Ade-GFP; control [CTRL]) or adenovirus-Cre-GFP (Ade-Cre-GFP; Dach1 KO). Representative Western blots of DACH1 protein expression in control and Dach1 KO tubule cells. GAPDH was used as loading control. (D) DACH1-ChIP-PCR for Ccnd1 and Ccnd2 in control kidney tubule cells. Three independent experiments were performed. (E) Integrated Genome Browser view of the human CCND2 locus. From top to bottom, the labels indicate the CCND2 locus, DACH1–ChIP-Seq, followed by human kidney snATAC-Seq in DCT, proximal tubule, loop of Henle, principal cell, intercalated cell. The box highlights the overlapping peaks between DACH1–ChIP-Seq and snATAC-Seq. (F) Relative gene expression of Ccna1, Ccnd1, Ccnd2, and Ccne1 in Ade-GFP or Ade-Cre-GFP transfected cells (n = 3). Gapdh was used as internal control. (G) Results of cell proliferation analysis using the MTT assay at 24 and 48 hours after Ade-GFP or Ade-Cre-GFP infection. (n = 3 in triplicate). *P < 0.05, **P < 0.01, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Binding Assay, Isolation, Infection, Control, Western Blot, Expressing, ChIP-sequencing, Gene Expression, Transfection, MTT Assay

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 9. DACH1 controls cell cycle in vivo. (A and B) Relative transcript expression of Ccnd2 (A) and Mki67 (B) in whole kidney samples of sham-treated and FA-injected WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/ Dach1fl/fl (Dach1 KO) mice. (C and D) Representative Ki-67 immunostaining (C) and quantification of Ki-67–positive cells per high-power field in kidney tissue of sham-treated and FA-inject- ed WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/Dach1fl/fl (Dach1 KO) mice. Scale bar: 20 μm. (E and F) Relative transcript levels of Ccnd2 (E) and Mki67 (F) in whole kidney samples of sham-treated and FA-injected WT and Pax8rtTA/TRE-Dach1 (Dach1 OE) mice. (A–D) Sham-treated group: WT (n = 4), Dach1 HZ (n = 4), Dach1 KO (n = 4); FA-treated group: WT (n = 10), Dach1 HZ (n = 8), Dach1 KO (n = 8). Light gray bars represent WT, red bars represent Dach1 HZ, and green bars represent Dach1 KO group. (E and F) Sham-treat- ed group: WT (n = 6), Dach1 OE (n = 6); FA-treated group: WT (n = 6), Dach1 OE (n = 8). Light gray bars represent WT,; red bars represent Dach1 OE. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: In Vivo, Expressing, Injection, Immunostaining

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 10. DACH1 transcriptionally controls cytokine expression in cultured kidney tubule cells. (A–C) DACH1-ChIP-qPCR for Ccl2 (A), Csf1 (B), Icam1 (C) using primary mouse kidney tubule epithelial cells. The y axis is presented as percentage of input (n = 3). (D) Integrated Genome Browser view of the human CSF1 locus. From top to bottom, the labels indicate CSF1 locus, DACH1-ChIP-seq, followed by human kidney snATAC-Seq (open chromatin region) in DCT, proximal tubule, loop of Henle, principal cells, intercalated cells. The red box highlights the overlapping peaks of DACH1 ChIP-Seq and human kidney snATAC-Seq. (E) The relative expression of Cxcl1, Cxcl2, Ccl2, Ccl5, Tnfa, Csf1, Il1b, Il17c, Il27, and Icam1 in Ade-GFP or Ade-Cre-GFP transfected renal tubule cells from Dach1fl/fl mice (n = 3). Gapdh was used as internal control. (F) Experimental scheme of Raw 264.7 macrophage chemotaxis assay. The supernatants of Ade-GFP or Ade- Cre-GFP-transfected renal tubule cells from Dach1fl/fl mice were added to the lower chamber. The number of macrophages (macrophage migration) was ana- lyzed by relative fluorescence units (RFU) after 2 or 24 hours (n = 3 in triplicate). *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Cell Culture, ChIP-qPCR, ChIP-sequencing, Transfection, Control, Chemotaxis Assay, Migration, Fluorescence

Journal: Journal of Clinical Investigation

Article Title: Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

doi: 10.1172/jci141801

Figure Lengend Snippet: Figure 11. DACH1 controls cytokine expression and macrophage infiltration. (A–C) Relative transcript expression of Ccl2 (A), Csf1 (B), and Icam1 (C) in kidneys of sham-treated and FA-treated WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/Dach1fl/fl (Dach1 KO) mice. (D–E) Representative immunostaining with F4/80 (macrophage marker) (D) and quantification of F4/80-positive area (E) in kidney tissue of sham-treated and FA-treated WT, KspCre/Dach1fl/WT (Dach1 HZ), and KspCre/Dach1fl/fl (Dach1 KO) mice. Scale bar: 20 μm. (F–H) Relative transcript expression of Ccl2 (F), Csf1 (G), and Icam1 (H) in kidneys of sham-treated and FA-treated WT and Pax8rtTA/TRE-Dach1 (Dach1 OE) mice. Gapdh was used as internal control. (A–D) Sham-treated group: WT (n = 10), Dach1 HZ (n = 10), Dach1 KO (n = 10); FA-treated group: WT (n = 10), Dach1 HZ (n = 8), Dach1 KO (n = 8). Light gray bars represent WT, red bars represent Dach1 HZ, and green bars represent Dach1 KO. (E and F) Sham-treated group: WT (n = 6), Dach1 OE (n = 6); FA-treated group; WT (n = 6), Dach1 OE (n = 8). Light gray bars represent WT; red bars represent Dach1 OE. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: DACH1 knockdown was performed using lentiviral expression vectors (pGFPC-shLenti) carrying shRNAs targeting DACH1 purchased from Origene (TL313572).

Techniques: Expressing, Immunostaining, Marker, Control

Journal: Head & face medicine

Article Title: Effect of DACH1 on proliferation and invasion of laryngeal squamous cell carcinoma.

doi: 10.1186/s13005-018-0177-1

Figure Lengend Snippet: Fig. 1 Representative DACH1 expression level in non-neoplastic tissues was lower than that in adjacent LSCC determined by IHC. a Representative DACH1 expression level in non-neoplastic tissues (upper images, × 200; lower image, × 400). b Representative DACH1 expression level in LSCC(upper images, × 200; lower image, × 400)

Article Snippet: Anti-DACH1 antibody was purchased from Origene.

Techniques: Expressing

Journal: Head & face medicine

Article Title: Effect of DACH1 on proliferation and invasion of laryngeal squamous cell carcinoma.

doi: 10.1186/s13005-018-0177-1

Figure Lengend Snippet: Fig. 2 (a) GFP plasmid-DACH1 expression in Hep-2 cells after 72 h. b No GFP expression in black Hep-2 cells. Fluorescence microscope images (× 200)

Article Snippet: Anti-DACH1 antibody was purchased from Origene.

Techniques: Plasmid Preparation, Expressing, Fluorescence, Microscopy

Journal: Head & face medicine

Article Title: Effect of DACH1 on proliferation and invasion of laryngeal squamous cell carcinoma.

doi: 10.1186/s13005-018-0177-1

Figure Lengend Snippet: Fig. 4 DACH1 inhibited proliferation and invasion of Hep-2 cells. a After plasmid-DACH1 transfection, the OD of the transfected group had no obvious increase as compared to the infected group at 72 h, P < 0.05. b After 72 h transfection, the number of invasive cells in each group. c The number of invasive cells in the black Hep-2 cells group. d The plasmid-DACH1 group, P < 0.05

Article Snippet: Anti-DACH1 antibody was purchased from Origene.

Techniques: Plasmid Preparation, Transfection, Infection

Journal: Head & face medicine

Article Title: Effect of DACH1 on proliferation and invasion of laryngeal squamous cell carcinoma.

doi: 10.1186/s13005-018-0177-1

Figure Lengend Snippet: Fig. 3 The expression level of DACH1 in two groups. a Real-time PCR analysis showed that DACH1 expression was significantly up-regulated after plasmid-DACH1 transfection, P < 0.05. b Western blot showed the protein level of DACH1 in transfected Hep-2 cells(1), and control Hep-2 cells (2)

Article Snippet: Anti-DACH1 antibody was purchased from Origene.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Western Blot, Control

Journal: Head & face medicine

Article Title: Effect of DACH1 on proliferation and invasion of laryngeal squamous cell carcinoma.

doi: 10.1186/s13005-018-0177-1

Figure Lengend Snippet: Fig. 5 Flow cytometric analysis of the effect of DACH1 on the cell cycle distribution in Hep-2 cells after transfection for 72 h. The G1 phase of black Hep-2 cells is significantly lower than plasmid-DACH1 cells, P < 0.05. a Cell cycle analysis of the blank control group Hep-2 cells. b Cell cycle analysis of DACH1-transfected Hep-2 cells

Article Snippet: Anti-DACH1 antibody was purchased from Origene.

Techniques: Transfection, Plasmid Preparation, Cell Cycle Assay, Control

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 2 DACH1 deletion PCa enhances AR signaling. A Interrogation of human PCa gene expression data [26], showing candidate genetic drivers ERG, ETV1/ETV4/FLI1, SPOP, FOXA1, and unknown. Samples with DACH1 homozygous (deep) genetic deletions (29/333) are shown as an additional subtype. The AR score (the average of the AR target gene expression) refers to a group of AR-responsive genes [26], and together with the expression Z-score of the AR target genes, are shown as colorimetric scales. The AR score-based gene names are shown. The androgen receptor (AR) activity, inferred by the induction of AR target genes, was increased in DACH1 homozygous (‘deep’) deletion PCa compared with normal (P = 2 × 10−5 by t-test) and ERG mutation groups (P = 0.003 by t-test). B AR mRNA and AR protein levels, shown for each DACH1 deletion sample, were not significantly different. C The iCluster [29], mRNA cluster, and SCNA (somatic copy-number alteration), and DNA methylation status are shown for the PCa classified by the corresponding gene deletion subtypes. D DACH1 homozygous deletions were enriched for iCluster 2 and 3 [29], mRNA cluster 2 (P = 0.0003 by Fisher exact test, SCNA (“more” somatic copy-number alteration, P = 0.0004 by Fisher exact test), but not for DNA methylation.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Gene Expression, Targeted Gene Expression, Expressing, Activity Assay, Mutagenesis, DNA Methylation Assay

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 3 Prostate-specific Dach1 gene deletion promotes prostate hyperplasia and dysplasia in OncoMice (15 weeks). A Schematic representation of transgenes integrated into mice. B Representative immunohistochemistry for Dach1, with data quantitated as mean ± standard error of the mean (SEM) for N = 20 (4 separate mice, with 5 views per mouse, in each group). C Blinded quantitative histology grading of prostate of multigenic mice at 15 weeks. Data are shown as mean ± SEM for N = 15 (5 separate mice, with 3 prostate areas [anterior, ventral, lateral] per mouse) in each group). H&E staining demonstrates the presence of a focal atypical intraductal proliferation in Dach1−/−prostate, compatible with prostatic intraepithelial neoplasia (PIN). Representative immunohistochemistry with results shown as mean ± SEM for Ki-67 (n = 20, 4 separate mice for each genotype, 5 views per mouse) (D), Beclin 1 (n = 9, 3 separate mice for each genotype, 3 views per mouse) (E); and AR (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 12 for Dach1fl/flmice, 3 separate mice, 2 views for one mouse and 5 views for other two mice) (F). Scale bars, 50 μm. A Student’s t test was performed for all comparisons.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Immunohistochemistry, Staining

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 4 Prostate-specific Dach1 gene deletion in TRAMP mice induces PIN lesions with increased TGFβ activity. Genome-wide expression analysis of TRAMP Dach1+/+ vs. Dach1−/−PIN lesions was analyzed for enrichment of known targets of upstream regulators using Ingenuity Pathway Analysis (IPA) and represented as (A) barplot was calculated by IPA activation Z-score labeled and as (B) bubble plot with size of the bubbles proportional to –log10 p values. C IHC was conducted for SMAD activation using SMAD2P, quantitated and shown as (D) mean ± SEM (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 10 for Dach1fl/flmice, 2 separate mice, 5 views per mouse). E–G Western blot of either PCa cell lines for the presence of DACH1 (E, F) or (G) TGFβ-treated (10 ng/ml for 24 h) PC3 cells illustrating induction of nuclear vimentin and cytoplasmic cyclin D1. Protein loading controls are β-tubulin (a marker of cytoplasmic proteins) and Lamin B1 (a marker for nuclear protein enrichment). H Microarray-based gene expression analysis of PC3 cells stably expressing DACH1, showing restraint of genes mediating TGFβ signaling (shown with blue arrows), including reduction of TGFB2 and TGFBR2 [33].

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Activity Assay, Genome Wide, Expressing, Activation Assay, Labeling, Western Blot, Marker, Protein Enrichment, Microarray, Gene Expression, Stable Transfection

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 6 DACH1 facilitates the recruitment of, and co-accumulates with, Ku70/Ku80 proteins at sites of DNA damage. A Co-accumulation of Ku-70/Ku-80 at laser micro irradiation-induced DSBs sites in Dach1+/+ 3T3 cells. B, C 24 h after transfection, the accumulation of DACH1 and Ku70/Ku80 in Dach1−/−3T3 cells transfected with EGFP or EGFP-tagged DACH1 and red fluorescent protein (RFP)-tagged Ku70 or RFP-tagged Ku80 expression vectors were treated with laser micro-irradiation (403 nm) to induce DSBs. Time is shown after micro-irradiation. Accumulation of the transfected proteins was indicated by EGFP (green) or RFP (red) fluorescence at laser-irradiated sites. Co-accumulation was visualized in yellow merged images. Time is shown in minutes and -fold increase in foci intensity is shown as mean ± SEM for N = 5 separate cells.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Irradiation, Transfection, Expressing

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: DACH1 marks crypt base cells in intestines and predicts poor outcomes in colorectal cancer patients. DACH1 expression in the mouse small intestine (a) and large intestine (b); scale bars=20 μm. In the mouse small intestine, DACH1 is expressed in crypt base cells interspersed between Paneth cells. Lysozymes (a marker of Paneth cells) were stained red, and DACH1 was stained green, which merged into cyan with DAPI (blue); scale bar=20 μm (c). DACH1 is also expressed in the human large intestine; scale bar=20 μm (d). DACH1 mRNA is overexpressed in colorectal cancer tissues compared to adjacent normal tissues, as detected by qRT-PCR (e), and IHC revealed that the expression of DACH1 increased in all stages of CRC when compared with the normal tissue (f) (* P < 0.05, ** P < 0.01, *** P < 0.001, Student's t-test; error bars: mean±SD). Elevated DACH1 expression at the invasive front of the tumour lesion; scale bar=100 μm (g). Kaplan-Meier survival analyses based on DACH1 expression for overall survival (h, P < 0.001, log-rank test, between high level and low level of DACH1) and disease-free survival (i, P < 0.001, log-rank test, between high level and low level of DACH1).

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Expressing, Marker, Staining, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: Baseline characteristics in patients with high and low expression of DACH1.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Expressing, Adjuvant

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: Univariate and multivariate analysis for overall survival.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Adjuvant

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: Univariate and Multivariate analyses of prognostic factors for disease-free survival.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Adjuvant

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: DACH1 suppression inhibits colon cancer cell growth in vitro and in vivo. The efficiency of DACH1 knockdown in colon cancer cell lines was confirmed by western blotting (a). Cells with knockdown of DACH1 displayed increased inhibition of cell growth compared with control cells (b and c, experiments were performed in triplicate). Representative images of xenografts at the end of the study (d) showing decreased growth in HCT116-shDACH1 cells compared with the control, including tumour volumes (e) and tumour weights (f). Knockdown of DACH1 significantly reduced the number of spheres formed by HCT116 cells (g and h, scale bar=200 μm). IHC assays showed weaker CD166, OLFM4 and SOX2 staining in xenografts in which DACH1 was knocked down than in the controls (i). For DACH1 staining images, the scale bars=50 μm, and for CD166, OLFM4 and SOX2 staining images, the scale bars=20 μm. For 2c, 2e, 2f and 2 g, * P < 0.05, ** P < 0.01, *** P < 0.001, Student's t-test. Error bars: mean±SD.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: In Vitro, In Vivo, Knockdown, Western Blot, Inhibition, Control, Staining

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: DACH1 stimulated CRC organoid formation. a. Representative images of the CRC organoid spheres. Sphere areas decreased in all the DACH1-knockout organoids (transfected with 3 single guide RNAs, sgRNAs, designated as sg1, sg2 and sg3) compared with that in the NT group. The scale bars for the upper row=100 μm, and the scale bars for the lower row=200 μm. b. DACH1 was efficiently knocked out in sgRNA-transfected organoids, as detected by WB. c. Total sphere areas decreased in the DACH1 sgRNA-transfected group compared with those in the NT group. d-e. The organoid areas in the presence of DACH1 expression were within the lower quartile more than the control; scale bars =200 μm. f-g. The average sphere areas of DACH1 knockout organoids were decreased compared with those in the NT control; scale bar=100 μm. h-i. The sphere growth rates in NT organoids were higher than those in the DACH1-knockout group; scale bar=100 μm. j-k. The organoid forming efficiency was decreased by DACH1 knockout. The scale bars in the left column=100 μm, and the scale bars in the right column=200 μm. For 3c, 3 g, 3i and 3k, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student's t-test. Error bars: mean±SD.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Knock-Out, Transfection, Expressing, Control

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: DACH1 overexpression enhances the growth of organoids derived from normal colon crypts and colorectal adenomas. DACH1 overexpression efficiency was verified by qRT-PCR (experiments were performed in triplicate) (a) and western blot (b). c. Overexpression of DACH1 enhanced the sphere formation of organoids derived from normal colon crypts (colonoid) compared with the control; scale bars=200 μm d. Overexpression of DACH1 significantly enhanced colonoid proliferation compared with the vector group. e. Overexpression of DACH1 significantly enhanced the colonoid formation rate compared with the control. DACH1 overexpression was verified by qRT-PCR (experiments were performed in triplicate) and western blot (f and g). h. Adenoma organoids with DACH1 overexpression expanded to almost twice the size of the control organoids; scale bars=200 μm. i. Overexpression of DACH1 significantly enhanced the proliferation of organoids derived from colorectal adenoma compared with the control. j. The adenoma organoid formation efficiency was improved by DACH1 overexpression. k-l. The organoid areas of DACH1-overexpressing organoids were within the lower quartile more than the control; scale bars=200 μm. m-n. The average adenoma organoid areas in DACH1-overexpressing organoids were significantly larger than those in the control; scale bars=100 μm. For 4a, 4d, 4e, 4f, 4i, 4j and 4n, * P < 0.05, ** P < 0.01, *** P < 0.001, Student's t-test. Error bars: mean±SD.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Over Expression, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation

Journal: EBioMedicine

Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

doi: 10.1016/j.ebiom.2020.102800

Figure Lengend Snippet: DACH1 promotes adenomas organoid formation via modulating BMP signalling. a DACH1 overexpression induced upregulation of cancer stem cell marker genes. b and c. Gene Ontology (GO) analysis showed that DACH1 overexpression induced the upregulation of stem cell signature genes and the downregulation of cell cycle arrest signature genes. d and e. DACH1 induced the downregulation of ATOH8, TGFβ3, MSX1, NBL1, BMP7, and FKBP4 and the upregulation of FKBP8, ID1, and MAPK3 (experiments were performed in triplicate). f. IF images showing the colocalization of SMAD4 and DACH1 in the nuclei; scale bars=20 μm g-h. DACH1 coprecipitated endogenous SMAD4. Reverse immunoprecipitation was confirmed with an anti-SMAD4 antibody. i. DACH1 overexpression increased the protein level of SMAD4 and decreased the level of phosphorylated SMAD4 (Thr276) [Thr277]. j and k. DACH1 knockdown led to an increase of the mRNA levels of NBL1 and BMP7 compared with the shNC group, and siRNA mediated SMAD4 knockdown in HCT116-shDACH1 cells eliminated the increase. l. DACH1 overexpression was sufficient to compensate for the withdrawal of Noggin and supported the formation of adenoma organoids. m. Addition of Noggin into the culture medium for 24 and 36 h decreased the mRNA levels of NBL1 and BMP7, which were increased by DACH1 knockdown in HCT116 cells. n and o. Overexpression of DACH1 upregulates LGR5, Notch1 and the protein level of NICD, while did not induce significant upregulation of HES1. Scale bars=20 μm. For 5d, 5e, 5k, 5 m and 5n, ** P < 0.01, * P < 0.05, Student's t-test. Error bars: mean±SD.

Article Snippet: For DACH1 staining, a CoraLite594-conjugated DACH1 antibody (Proteintech, Cat. CL594-60082, 1:200) was used.

Techniques: Over Expression, Marker, Immunoprecipitation, Knockdown