Additional file 1 of Systematic in vivo candidate evaluation uncovers therapeutic targets for LMNA dilated cardiomyopathy and risk of Lamin A toxicity
Creators: name: Tan, Chia Yee
name: Chan, Pui Shi
name: Tan, Hansen
name: Tan, Sung Wei
name: Lee, Chang Jie Mick
name: Wang, Jiong-Wei
name: Ye, Shu
name: Werner, Hendrikje
name: Loh, Ying Jie
name: Lee, Yin Loon
name: Ackers-Johnson, Matthew
name: Foo, Roger S. Y.
name: Jiang, Jianming
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology

description: Additional file 1: Figure S1. Upregulation of Lamin A and Lamin C in Lmna DCM mice. Evaluation of the upregulated level of Lamin A and Lamin C in vivo by western blot in mouse whole heart tissue lysates of control or Lmna DCM groups with control, Lamin A or Lamin C upregulation. Figure S2. Upregulation of Lamin A in mice. (a) Experimental timeline showing timepoints of virus injection and echocardiogram. Cardiac performance was assessed by echocardiogram at 5.5 weeks-old. SR and H&amp;E staining of paraffin heart sections 3-4 weeks after transduction of EGFP control or Lamin A. Quantification of myocardial fibrosis of SR sections, virus dose, 1.0E+13 vg/kg, n = 5, Mann-Whitney test. For complete heart images: magnification = 4 ×, scale bar =1000 µm; for enlarged images: magnification = 20 ×, scale bar = 100 µm. (b, c) Quantitative real-time PCR analyses of Nppa, Nppb, Col1a1 and Col1a2 in mice transduced with EGFP control or Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction and Mann-Whitney test. Figure S3. Upregulation of mature Lamin A by AAV leads to DCM and cardiac fibrosis. (a) SR and H&amp;E staining of paraffin heart sections and quantifications of mice transduced with EGFP control or mature Lamin A. Quantification of myocardial fibrosis of SR sections, virus dose, 2.0E+13 vg/kg, n = 5, two-tailed, unpaired T-test with Welch correction. For complete heart images: magnification = 4 ×, scale bar =1000 µm; for enlarged images: magnification = 20 ×, scale bar = 100 µm. (b, c) Quantitative real-time PCR analyses of Nppa, Nppb, Col1a1 and Col1a2 in mice transduced with EGFP control or mature Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction. (d) Western blot of p-Smad2 protein levels in mouse heart tissues of mice transduced with EGFP control or mature Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction. (e–g) Paraffin heart sections (left) and quantifications (right) of (e) αSMA (red), (f) Iba-1 (red), (g) CD3 (red), cTnI (green) and DAPI (blue) positive cells in mice after transduction of EGFP control or mature Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction, scale bar = 50 µm. Figure S4. DNA damage levels in Lamin A DCM. (a) Representative images (left) and quantifications (right) of paraffin heart sections with immunostaining with for γH2AX (red), cTnI (green) and DAPI (blue), n = 5, two-tailed, unpaired T-test with Welch correction, scale bar = 5 µm. Arrow indicates γH2AX-positive CMs in Lamin A DCM. Figure S5. Expression levels of selected candidates in Lmna DCM. Quantitative real-time PCR analyses of Tgfb1, Smad2, Smad3, Atp2a2, Fgf16, Mapk14, Sun1, Bmp7, Ctgf, Yy1, Yap1 and Rptor expressions in control or Lmna DCM mice, n = 5, two-tailed, unpaired T-test with Welch correction and Mann-Whitney test. Figure S6. Knockdown efficacy of RNAi in vitro. 2-color system and quantification of shRNA knockdown efficacy of selected candidates. HEK293T cells co-transfected with selected candidates (green) and corresponding shRNAs or control shRNA (Red), n=4, two-tailed, unpaired T-test with Welch correction. Magnification = 4 ×, scale bar=500 µm. Figure S7. Evaluation of Survival Rate for selected candidates in wildtype mice. Survival curve of wildtype mice supplemented with Ctrl shRNA (black), EGFP Ctrl (black) or selected candidates (red), n = 10. Figure S8. Upregulation level and RNAi efficacy in vivo for positive candidates. Evaluation of the upregulated level of positive candidates Yy1, Bmp7 and aYAP1 and RNAi knockdown efficacy of positive candidates Smad3, Ctgf and Sun1 in vivo by western blot in mouse heart tissues of control or Lmna DCM groups. * indicates a non-specific band. Figure S9. Different dose of AAV9-shRNA. (a and b) Paraffin heart sections of (left) of (a) Sun1 (red), (b) Yap1 (red), cTnI (green) and DAPI (blue) and quantifications (right) of (a) Sun1 intensity (red), (b) Yap1 intensity (red) after transduction of 1E+13 vg/kg or 2E+13 vg/kg dose for (a) Sun1 shRNA or (b) Yap1 shRNA in control or Lmna DCM mice, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction, scale bar = 10 µm. Arrows indicate Sun1 (a) or Yap1 (b) positive CMs. Figure S10. Different dose of AAV9 overexpression. (a and b) Paraffin heart sections of (left) (a) Yap1 (red), (b) Yy1 (red), cTnI (green) and DAPI (blue) and quantifications (right) of a aYAP1 intensity (red), b Yy1 intensity (red) after transduction of 1E+13 vg/kg or 2E+13 vg/kg dose for (a) aYAP1 or (b) Yy1 in control or Lmna DCM mice, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction, scale bar = 10 µm. Arrows indicate Yap1 (a) or Yy1 (b) positive CMs. Figure S11. Long-term cardiac performance of Lmna DCM supplemented with Lamin C or Sun1 shRNA. (a and b) Evaluation of long-term cardiac performance by echocardiography in Lmna DCM mice supplemented with (a) Lamin C or (b) Sun1 shRNA, n = 10. Figure S12. Sun1 distribution and nuclear protrusion in Lmna DCM. (a) Western blot of Sun1 protein levels in mouse heart tissues of control or Lmna DCM mice. (b) Paraffin heart sections of (left) of Sun1 (red), cTnI (green), Lamin A/C (teal) and DAPI (blue) and quantifications (right) of abnormal Sun1 distribution and nuclear protrusion in Lmna DCM, n=5, two-tailed, unpaired T-test with Welch correction. Arrows indicate Sun1 positive CMs, white arrows indicate abnormal Sun1 distribution in CMs. (c) Paraffin heart sections (left) of PCM1 (red), cTnI (green) and DAPI (blue) and quantifications (right) of nuclear protrusion in control or Lmna DCM mice supplemented with control or Sun1 shRNA, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction, scale bar = 10 µm. Figure S13. Nesprin1 distribution and nuclear shape in Lmna DCM. (a) Quantitative real-time PCR analyses of Syne1 (Nesprin1) in control or Lmna DCM mice, n = 5, two-tailed, unpaired T-test with Welch correction. (b) Western blot of Nesprin1 protein levels in mouse heart tissues of control or Lmna DCM mice. (c) Paraffin heart sections of Sun1 (red), Nesprin1 (teal), cTnI (green) and DAPI (blue) positive cells, scale bar = 10 µm. (d) Paraffin heart sections of (left) of Nesprin1 (red), cTnI (green) and DAPI (blue) and quantifications (right) of abnormal Nesprin1 distribution and nuclear protrusion in Lmna DCM mice, n = 5, two-tailed, unpaired T-test with Welch correction, scale bar = 10 µm. Arrows indicate Nesprin1 positive CMs, white arrows indicate abnormal Nesprin1 distribution in CMs. Figure S14. KASH domain suppresses Lmna DCM and cardiac fibrosis. (a and b) SR and H&amp;E staining of paraffin heart sections and quantifications of Lmna DCM mice supplemented with control or KASH domain. Quantification of myocardial fibrosis of SR sections, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction. For complete heart images: magnification = 4 ×, scale bar =1000 µm; for enlarged images: magnification = 20 ×, scale bar = 100 µm. (c) Quantitative real-time PCR analyses of Nppa, Nppb, Col1a1 and Col1a2, KASH domain in Lmna DCM mice supplemented with control or KASH domain, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction. Figure S15. Effect of DNSUN1 on inducible Lmna DCM mice. (a) Survival curve of inducible Lmna DCM mice treated with DNSUN1 (Red) at a dose of 1E+14 vg/kg compared to inducible Lmna DCM mice (Black) and Ctrl (Blue), n ≥ 5, Log- rank (Mantel- Cox) test. Figure S16. LMNA Mutations. Schematic diagram of LMNA mutations depicted by green lines located along the Prelamin A and Lamin A/C. Diagram is adapted from Broers et al [63].