Journal: bioRxiv

Article Title: AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

doi: 10.64898/2025.12.21.695613

Figure Lengend Snippet: a , Schematic representation of the generated transgenic animals and the expected outcome after tamoxifen induction. b , Representative images of ctrl and dnAXL heart sections showing mosaic pattern of endogenous GFP+ (green, non-converted), endogenous mCherry+ (red, converted in ctrl) and stained Flag-tag (red, converted in dnAXL). Squares show magnified regions stained with EdU showing cycling CMs. Yellow arrowheads show converted EdU+ CM. White arrowheads show non-converted EdU+ CM. c , HCR staining for Axl showing no expression in non-converted CMs while having high expression in adjacent cells. d , Quantification of EdU+ converted CMs. e - i HCR staining and quantification of number of positive cells for known border zone markers Nppa ( e -g) and Ankrd1 ( h,i ) of dnAXL and ctrl hearts. j - m , Mason’s trichrome staining on ctrl ( j ) and dnAXL ( k ) heart sections, 8 weeks post cryo injury showing the damage area and residual fibrotic region (pale color and blue region, marked by red dashed line). l , m, quantification of damage area as taken by the 3 sections which showed the largest damage area. Data is presented as damage area relative to size of ventricle ( l ) and average area size ( m ). * P < 0.05, Statistical test in all data: Mann-Whitney one-tailed T-test.

Article Snippet: For immunostaining, the cells were incubated overnight at 4°C with the following antibodies diluted in the blocking solution: AXL (1:100, PA5-106118, ThermoFisher), Alpha actinin (1:300, ab9465, abcam), GFP (1:200, R1091P, Origene).Cells were then washed three times with PBS and stained for 60 min at room temperature with suitable secondary antibody, diluted 1:500 in PBS-Triton 0,1%, and DAPI 1:1000 (4,6-diamidino-2-phenylindole dihydrochloride).

Techniques: Generated, Transgenic Assay, Staining, FLAG-tag, Expressing, MANN-WHITNEY, One-tailed Test

Journal: bioRxiv

Article Title: AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

doi: 10.64898/2025.12.21.695613

Figure Lengend Snippet: a , Representative images of P1 neonatal CMs 5 days after AAV9-AXL or AAV9-GFP infection stained for AXL or GFP (magenta), linage labeling of CMs (green), α-actinin (gray) and dapi. b , Representative images same as ( a ) only with addition of 0uM, 0.1uM or 1uM R428 (axl inhibitor). c , Quantification of sarcomere integrity from a,b. d-h , in-vivo AAV9-AXL/GFP administration at neonatal P1 and analyzed at 8 weeks. Representative image of AXL induced sarcomere disassembly ( d ) as evident by less α-actinin staining as calculated by automated sarcomere detection per area (average between samples in e , all cells combined in f ) and lower mean α-actinin mean pixel intensity (average between samples in g , all cells combined in h ). n=504 AXL-cells and 709 AXL+ cells. i, Representative images and quantification of EdU cell cycle indicators of P1 CMs in-vitro, 3 days after addition of AAV9-AXL or AAV9-GFP. j , Volcano plot generated from bulk-RNA seq of 6xAAV9-AXL and 4xAAV9-GFP samples, of P1 CMs in-vitro , 3 days after infection. Genes in red are significantly up or downregulated (1.5 > log2FC, -1.5 < log2FC, -log10P < 1xe-20), as calculated by DESeq2. Gene in blue are significant but not sufficiently changed. Genes in green are sufficiently up or down regulated but not significantly. k , Heatmap of known border zone or remote zone markers in AAV9-AXL (AXL) or AAV9-GFP (GFP) taken from bulk RNA-seq. Asterisk next to gene name shows p<0.05 using DESeq2 statistical test. m , Heatmap of known glycolytic enzymes in AAV9-AXL (AXL) or AAV9-GFP (GFP) taken from bulk RNA-seq. Asterisk next to gene name shows p<0.05 using DESeq2 statistical test. m , Ratio of Extracellular acidification rate (ECAR)/ Oxygen consumption rate (OCR) indicating the preference of pyruvate fate of either fermentation or oxidation by mitochondria. Glycolysis induction was calculated by subtraction of values after 2-Deoxy-D-glucose induction from glucose induction values. n , Heatmap of major ion channels selected from expression levels of adult mouse hearts in AAV9-AXL (AXL) or AAV9-GFP (GFP) taken from bulk RNA-seq. Asterisk next to gene name shows p<0.05 using DESeq2 statistical test. o , Myh6/Myh7 gene expression ratio as calculated from bulk RNA-seq normalized counts. p , Myl2/Myl7 gene expression ratio as calculated from bulk RNA-seq normalized counts. In all relevant panels * P < 0.05, ** P < 0.01, *** P < 0.001 ****P<0.0001 (statistical test: Mann-Whitney two-tailed T-test). In panels k,l and n, asterisk shows significant change as calculated by DESeq2.

Article Snippet: For immunostaining, the cells were incubated overnight at 4°C with the following antibodies diluted in the blocking solution: AXL (1:100, PA5-106118, ThermoFisher), Alpha actinin (1:300, ab9465, abcam), GFP (1:200, R1091P, Origene).Cells were then washed three times with PBS and stained for 60 min at room temperature with suitable secondary antibody, diluted 1:500 in PBS-Triton 0,1%, and DAPI 1:1000 (4,6-diamidino-2-phenylindole dihydrochloride).

Techniques: Infection, Staining, Labeling, In Vivo, In Vitro, Generated, RNA Sequencing, Expressing, Gene Expression, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

doi: 10.64898/2025.12.21.695613

Figure Lengend Snippet: a,b, Representative images of AAV9-AXL (a) or AAV9-GFP (b) treated cardiac cultures depicting sarcomere segmentation overlayed on a-actinin and AXL or GFP staining. c, representative image of non-AXL OE cell next to AXL OE cell (green) showing disassembled sarcomere structure. d,e, Quantification of sarcomere coverage in AXL/GFP OE cells. Sarcomere detection was done in an automated manner and assayed on positive cells detected by Cellpose. Data shown as area/cell ( d ) or fold change relative to GFP ctrl as calculated from a ( e ). Data are presented as average ± s.e.m. * P < 0.05, ** P < 0.01 (statistical test: Mann-Whitney two-tailed T-test).

Article Snippet: For immunostaining, the cells were incubated overnight at 4°C with the following antibodies diluted in the blocking solution: AXL (1:100, PA5-106118, ThermoFisher), Alpha actinin (1:300, ab9465, abcam), GFP (1:200, R1091P, Origene).Cells were then washed three times with PBS and stained for 60 min at room temperature with suitable secondary antibody, diluted 1:500 in PBS-Triton 0,1%, and DAPI 1:1000 (4,6-diamidino-2-phenylindole dihydrochloride).

Techniques: Staining, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

doi: 10.64898/2025.12.21.695613

Figure Lengend Snippet: a , Principal component analysis (PCA) of AAV9-AXL or AAV9-GFP infected, bulk RNA-seq samples. b , IPA analysis of most activated and inhibited GO terms. c , Quantification of cell death of CM by Tunel assay after norm-oxic and hypoxic conditions. statistical test: Mann-Whitney two-tailed T-test . d , KEGG enriched go terms based on the bulk RNA-seq results depicted in ( a )

Article Snippet: For immunostaining, the cells were incubated overnight at 4°C with the following antibodies diluted in the blocking solution: AXL (1:100, PA5-106118, ThermoFisher), Alpha actinin (1:300, ab9465, abcam), GFP (1:200, R1091P, Origene).Cells were then washed three times with PBS and stained for 60 min at room temperature with suitable secondary antibody, diluted 1:500 in PBS-Triton 0,1%, and DAPI 1:1000 (4,6-diamidino-2-phenylindole dihydrochloride).

Techniques: Infection, RNA Sequencing, TUNEL Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

doi: 10.64898/2025.12.21.695613

Figure Lengend Snippet: a , Heatmap depicting glycolysis related gene expression levels. b , Heatmap showing all mitochondrial genes from the 5 energy transfer complexes. c , Heatmap showing fatty acid oxidation enzymes in GFP or AXL overexpression. Red is increased while blue means downregulated. d, Ratio of Extracellular acidification rate (ECAR)/ Oxygen consumption rate (OCR) in the Cell Mito Stress kit, indicating the preference of pyruvate fate of either fermentation or oxidation by mitochondria. Values show either basal levels or with inhibition of mitochondria using FCCP. *** P < 0.001 (statistical test: Mann-Whitney two-tailed T-test for basal and FCCP treatment individually).

Article Snippet: For immunostaining, the cells were incubated overnight at 4°C with the following antibodies diluted in the blocking solution: AXL (1:100, PA5-106118, ThermoFisher), Alpha actinin (1:300, ab9465, abcam), GFP (1:200, R1091P, Origene).Cells were then washed three times with PBS and stained for 60 min at room temperature with suitable secondary antibody, diluted 1:500 in PBS-Triton 0,1%, and DAPI 1:1000 (4,6-diamidino-2-phenylindole dihydrochloride).

Techniques: Gene Expression, Over Expression, Inhibition, MANN-WHITNEY, Two Tailed Test