Journal: bioRxiv

Article Title: Targeting lysosomal damage is a new therapeutic perspective for Duchenne Muscular Dystrophy

doi: 10.1101/2025.02.11.637610

Figure Lengend Snippet: ( a) Schematic representation of study set-up. 6-week-old Dmd mdx mice were inject intravenously with a rAAV encapsulating a µ-dystrophin encoding sequence under the control of spc512 promoter, at 2 doses of 5e12 vg/kg (low dose) and 1e13 vg/kg (high dose). ( b) Histological characterization of GA muscle. Top panel: Dystrophin staining of GA cross-sections (DYSB antibody recognizes N-terminal part of dystrophin). Middle Panel: Sirus Red labeling of GA cross-sections. Bottom panel: Filipin (free cholesterol) and Wheat Germ Agglutinin (WGA) (labeling of nuclear and sarcolemma membranes) labeling of GA cross-sections. ( c) Viral copy numbers (VCN) quantification in GA muscle. Data are presented as Scatter plot (average ± SD) of relative abundance to Rplp0. ( d) Quantification of µ-dys transcripts in GA muscle. Data are presented as Scatter plot (average ± SD) of relative abundance to Rplp0. ( e) Quantification of dystrophin positive fibers in GA muscle (The antibody used, DYSB, recognize the N-terminal of the µ-dys protein as well as the WT murine dystrophin). ( f) Dosage of CK in serum of mice 6-weeks post-injection. Data are presented as scatter plot (average ± SD). ( g) ELISA quantification of MYOM3 in the serum of the mice 6-weeks post injection. Data are presented scatter plot (average ± SD). ( h) Global force evaluation by an escape test before sacrifice of mice. Data are presented as scatter plot (average ± SD) of global force normalized to mice body weight. ( i) Quantification of fibrosis in the GA (Sirus red positive area) (n=6, two sections quantified per mouse). ( j) Scatter plot (average ± SD) displaying free cholesterol quantification in whole GA muscle lysates (n=5). ( k) Representative confocal images of transversal sections of GA muscle immunostained for LAMP2 (green) and LGALS3 (red). ( l) Relative Lgals3 mRNA expression in the GA normalized to Rplp0. Scatter plots represent average ± SD (n=6). ( m) Quantification of double positive puncta (LAMP2+LGALS3+) in the myofibers of GA muscles. Data are represented as scatter plots (average ± SD). ( n ) Western blot analysis of GA muscles (n=3) for LGALS3. GAPDH is used as loading control. ( o) Band intensity quantification of LGALS3 relative to GAPDH. Data are represented as scatter plot of average ± SD (n=3). An ANOVA test with Tukey multiple comparison, or a Kruskal-Wallis with Dunn’s multiple comparisons (when values didn’t follow a normal distribution), were used for statistical comparisons. p-value *p<0.05, **p<0.05, ***p<0.001, ****p<0.0001, ns=non-significant. Grey Asterix represents relative comparison to ‘WT+Phy Ser’ group, red Asterix represent comparison to ‘mdx+Phy Ser’ group.

Article Snippet: Anti-MYOM3 polyclonal capture antibody (Proteintech Lab, ref. 17692-1-AP) was coated to the wells of a multi-array plate containing electrodes (MSD, ref. L15XA-3) (overnight, 4°C).

Techniques: Sequencing, Control, Staining, Labeling, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Muscles, Western Blot, Comparison

Journal: bioRxiv

Article Title: Targeting lysosomal damage is a new therapeutic perspective for Duchenne Muscular Dystrophy

doi: 10.1101/2025.02.11.637610

Figure Lengend Snippet: (a) Schematic representation of study set-up. 4-week-old Sgcg -/- mice were injected intravenously with a rAAV9 encapsulating the human sequence of SGCG, under the control of a desmin promoter, at a dose of 2e13 vg/kg. ( b) Histological characterization of GA muscle. Top panel: representative images of GA cross-sections immunostained with antibody specific for the human SGCG. Bottom panel: representative images of GA cross-sections labeled with HES. ( c) Western blot analysis of GA muscles for the expression of human SGCG. GAPDH is used as loading control. ( d) Viral copy number (VCN) quantification in the GA muscle. Data are presented as scatter plot (average ± SD) (n=10, right and left GA muscles are analyzed). ( e) Relative SGCG mRNA expression in the GA normalized to Rplp0. Scatter plots represent average ± SD. ( f) Dosage of CK in serum of mice 4-weeks post-injection. Data are presented as scatter plot (average ± SD). ( g) ELISA quantification of MYOM3 in the serum of the mice 4-weeks post injection. Data are presented scatter plot (average ± SD). ( h) Global force evaluation by an escape test before sacrifice of mice. Data are presented as scatter plot (average ± SD) of global force normalized to mice body weight. ( i) Relative Lgals3 mRNA expression in the GA normalized to Rplp0. Scatter plots represent average ± SD. ( j) Quantification of double positive puncta (LAMP2+LGALS3+) in the myofibers of GA muscles. Data are represented as scatter plots with median (n>338). ( k) Representative confocal images of transversal sections of GA muscle immunostained for LAMP2 (green) and LGALS3 (red). An ANOVA test with Tukey multiple comparison was used for all statistical comparisons. ANOVA p-value *p<0.05, **p<0.05, ***p<0.001, ****p<0.0001, ns=non-significant.

Article Snippet: Anti-MYOM3 polyclonal capture antibody (Proteintech Lab, ref. 17692-1-AP) was coated to the wells of a multi-array plate containing electrodes (MSD, ref. L15XA-3) (overnight, 4°C).

Techniques: Injection, Sequencing, Control, Labeling, Western Blot, Muscles, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: Targeting lysosomal damage is a new therapeutic perspective for Duchenne Muscular Dystrophy

doi: 10.1101/2025.02.11.637610

Figure Lengend Snippet: ( a ) Schematic representation of study set-up. Dmd mdx-4Cv mice were started on trehalose treatment (2% dilution in drinking water) at 3-weeks. For Group 4 and 5, mice were injected intravenously with a rAAV9 encapsulating the µ-dys sequence at a 7e12 vg/kg dose. ( b ) Histological characterization of muscles. Top panel: H&E labeling of GA muscle cross-sections. Middle Panel: Sirius red labeling of diaphragm and GA muscle cross-sections. Bottom Panel: Representative images of GA muscle serial cross sections immunostained for mouse immunoglobulin (IgG), laminin and CD11b. ( c ) Dosage of CK in serum of mice before escape test. Data are presented as scatter plot (average ± SD, n=5). ( d ) ELISA quantification of MYOM3 in the serum of the mice before escape test. Data are presented scatter plot (average ± SD, n=5). ( e ) Global force evaluation by an escape test before sacrifice of mice. Data are presented as scatter plot (average ± SD, n=5) of global force normalized to mice body weight (N/g). ( f ) Analysis of myofiber size distribution in the GA muscle. Data presented as box plots (Median + minimum/maximum) show variance coefficient calculated as [standard deviation of the muscle fiber size/mean of muscle fiber size] *1000. (g) Centronucleation Index in the GA muscle. Data are presented as box plots (Median + minimum/maximum). h-i) Fibrosis analysis of GA and diaphragm, done by quantification of Sirius red positive area on transversal sections. Data are presented as scatter plots (average ± SD). ( j ) Quantification of permeabilized fibers positive for mouse IgG staining. Data are presented as scatter plot (average ± SD) of number of positive fibers normalized to muscle cross-sectional area. ( k ) Evaluation of CD11b+ cells infiltration in the muscle, done by quantifying CD11b area normalized to muscle cross-sectional area. Data are presented as a scatter plot (average ± SD). An ANOVA test with Tukey multiple comparison was used for statistical comparisons (compared to WT control and mdx control). ANOVA p-value *p<0.05, **p<0.05, ***p<0.001, ****p<0.0001, ns=non-significant. Black Asterix represents relative comparison to ‘WT control’ group, red Asterix represent comparison to ‘mdx control’ group. An unpaired two-tailed t.test was used for statistical comparisons between mdx-µ-dys and mdx_µ-dys+TR groups. T.test p-value *p < 0.05, **p<0.01.

Article Snippet: Anti-MYOM3 polyclonal capture antibody (Proteintech Lab, ref. 17692-1-AP) was coated to the wells of a multi-array plate containing electrodes (MSD, ref. L15XA-3) (overnight, 4°C).

Techniques: Injection, Sequencing, Muscles, Labeling, Enzyme-linked Immunosorbent Assay, Standard Deviation, Staining, Comparison, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

doi: 10.1101/2023.10.02.560523

Figure Lengend Snippet: CETSA® thermal shifts (Δ T m ) for RIPK1 with Takeda compound 22 in human whole blood from multiple healthy donors. Measurements with (A) Alpha CETSA® or (B) MSD CETSA®. Comparability of CETSA® melting and shift curves with and without a freeze-thaw pause step for (C,D) Alpha CETSA® and (E,F) MSD CETSA®. (G) Comparison of compound 22 Δ T m variability between Alpha CETSA® (blue) and MSD CETSA® (red) assays across multiple, different donors; between different sets of donors analyzed with MSD CETSA® on different days (red and green); and the same donor sample set analyzed using MSD CETSA® without (green) or with (blue) a freeze-thaw pause step.

Article Snippet: MSD multiarray 96-well plates (Meso Scale Discovery L15XA-3) were coated overnight at 4 °C with 300 ng/mL mouse anti-human RIPK1 capture antibody (1:100 dilution in PBS of BD Bioscience BD610459 lot 0314064, 50 µL/well).

Techniques: Comparison

Journal: bioRxiv

Article Title: Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

doi: 10.1101/2023.10.02.560523

Figure Lengend Snippet: CETSA® formats for cells and whole blood. (A) Schematic for CETSA® with immunoblot ( top ) or AlphaLISA and MSD detection ( bottom ). (B) Simple Western CETSA® signal for soluble RIPK1 in Jurkat cells after treating with 10 μM Takeda compound 22 or vehicle and heating. The thermostable protein SOD1 is also probed as a loading control. (C) Simple Western quantification of RIPK1 CETSA® melting curves in Jurkats. Data are relative to the SOD1 intensity at each temperature and normalized to RIPK1 signal for vehicle at 37°C. (D) Immunoblot CETSA® signal for soluble RIPK1 in whole blood after treating ex vivo with 10 μM compound 22, heating, and isolating PBMCs. (E) MSD CETSA® and (F) Alpha CETSA® with whole blood with 10 μM compound 22 and vehicle. (G) Alpha CETSA® data for isolated PBMCs. Data for B, C, D are representative of two biological replicates. 1A was created with Biorender.com.

Article Snippet: MSD multiarray 96-well plates (Meso Scale Discovery L15XA-3) were coated overnight at 4 °C with 300 ng/mL mouse anti-human RIPK1 capture antibody (1:100 dilution in PBS of BD Bioscience BD610459 lot 0314064, 50 µL/well).

Techniques: Western Blot, Ex Vivo, Isolation

Journal: bioRxiv

Article Title: Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

doi: 10.1101/2023.10.02.560523

Figure Lengend Snippet: Concentration-response (CR) CETSA® with whole blood. (A) CETSA® melting curves of whole blood with MSD CETSA® at 6 different concentrations of Takeda compound 22 or vehicle. CR CETSA® stabilization of RIPK1 by compound 22 with (B) MSD CETSA® in whole blood from four healthy donors at 53 °C or (C) Alpha CETSA® in whole blood from six donors at 50 °C. Comparison of active and inactive enantiomers GSK’963 and GSK’962 with whole blood from multiple donors with (D) MSD CETSA® or (E) Alpha CETSA®.

Article Snippet: MSD multiarray 96-well plates (Meso Scale Discovery L15XA-3) were coated overnight at 4 °C with 300 ng/mL mouse anti-human RIPK1 capture antibody (1:100 dilution in PBS of BD Bioscience BD610459 lot 0314064, 50 µL/well).

Techniques: Concentration Assay, Comparison

Journal: bioRxiv

Article Title: Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

doi: 10.1101/2023.10.02.560523

Figure Lengend Snippet: Correlations for engagement measurements across multiple RIPK1 inhibitors. Comparisons for (A) Alpha CETSA® and MSD CETSA® and (B) MSD CETSA® and the TEAR1 assay.

Article Snippet: MSD multiarray 96-well plates (Meso Scale Discovery L15XA-3) were coated overnight at 4 °C with 300 ng/mL mouse anti-human RIPK1 capture antibody (1:100 dilution in PBS of BD Bioscience BD610459 lot 0314064, 50 µL/well).

Techniques: