Journal: bioRxiv

Article Title: The Onset of Breathing at Birth Switches Off Regenerative Capacity

doi: 10.64898/2026.10.03.756425

Figure Lengend Snippet: a-c, Representative images of phosphorylated S6 (p-S6, green), RBPMS (red), and DAPI (blue) staining in whole retinal sections (left), with magnified views (middle), and normalization of p-S6/RBPMS intensity (right) from pre-birth (E18.5) embryos, 5 min, and 12 hr postnatal pups. Scale bars, 200 μm (whole retinal section); 50 μm (magnified views). d, Quantification showing that the percentage of p-S6-positive RGCs significantly decreases after birth. Each circle represents the mean percentage of p-S6-positive RGCs among total RBPMS-positive RGCs in the ganglion cell layer (GCL) of each retina. For each whole retina section, all RGCs were included in the quantification. ****p < 0.0001; N = 4, 3 and 3 biological replicates for pre-birth, 5 min post-birth and 12 hr post-birth groups, respectively; unpaired Welch’s t-test. e, Western blot showing that mTOR activity markers (p-S6, p-S6K, and p-4EBP1) decrease markedly in brain, heart, and hindlimb muscles within 5 min after birth. Quadriceps femoris muscles and biceps femoris muscles from both left and right hindlimbs were pooled together to generate hindlimb muscles sample for each mouse. f, Quantification of Western blot result shown in (e) . *p < 0.05, **p < 0.01, ***p < 0.001; N = 3 and 4 biological replicates for pre-birth and 5 min post birth, respectively; unpaired Welch’s t-test. g, Experimental scheme (left) illustrating conditions with deprivation of maternal nutrient supply or inhibition of breathing onset. Representative images (right) of p-S6, RBPMS, and DAPI staining in whole retinal sections (from newborns at 5 min after birth) and corresponding magnified views under the indicated conditions. 200 μm (whole retinal section); 50 μm (magnified views). h, Quantification showing the percentage of p-S6-positive RGCs under different conditions. At 5 min after birth, deprivation of maternal nutrient supply does not alter p-S6 levels, whereas initiation of breathing significantly decreases mTOR activity. ***p < 0.001; n.s., not significant; N = 5, 3, 3 and 3 biological replicates for (“+ nutrient - breathing”), (“- nutrient - breathing”), (“+ nutrient + breathing”) and (“- nutrient + breathing”) groups, respectively; unpaired Welch’s t-test.

Article Snippet: Primary antibodies against phospho-S6 (Ser235/236, CST 4858S), phospho-S6K (Thr389, CST 9234T), phospho-4EBP1 (Thr37/46, CST 2855T), total S6 (CST 2217T), S6K (CST 9202S), 4EBP1(CST 9644T) and actin (CST 4970T) were obtained from Cell Signaling Technology.

Techniques: Staining, Western Blot, Activity Assay, Muscles, Inhibition

Journal: bioRxiv

Article Title: The Onset of Breathing at Birth Switches Off Regenerative Capacity

doi: 10.64898/2026.10.03.756425

Figure Lengend Snippet: a, Experimental scheme for intravitreal injection of AAV2-mSncg-Cre or GFP control virus and optic nerve crush in Snrk fl/fl mice. b, Representative images of phosphorylated S6 (p-S6, green) and RBPMS (red) staining in the retina section of Snrk fl/fl mice injected with either GFP or Cre-expressing AAVs. Scale bar, 20 µm. c, d, Representative full thickness optic nerve images showing CTB-labeled axons at 14 days after injury in Snrk fl/fl mice injected with AAV-GFP (c) or AAV-Cre (d) . Scale bar, 100 µm. The red asterisk indicates the ONC site. e, Quantification of CTB-labeled fluorescent intensity distal to the crush site in Snrk fl/fl mice injected with AAV-Cre or AAV-GFP. Data are shown as mean ± SEM; N = 7 and 8 biological replicates for AAV-GFP and AAV-Cre injected mice, respectively. ***p < 0.001; mixed-effects analysis. f, Representative images of phosphorylated S6 (p-S6, green) and RBPMS (red) staining in the retinal section of pre-birth (E18.5) embryos and 5 min post-birth newborns of wild-type or Snrk cKO. Scale bar, 20 µm. g, Quantification showing the percentage of p-S6-positive RGCs. Data are presented as mean percentages of p-S6-positive and RBPMS-positive cells among total RBPMS-positive cells in the GCL of each retina. ***p < 0.001; n.s., not significant; N = 4 wild-type and 5 Snrk cKO biological replicates for pre-birth; N = 5 wild-type and 4 Snrk cKO biological replicates at 5 min post birth; unpaired Welch’s t-test.

Article Snippet: Primary antibodies against phospho-S6 (Ser235/236, CST 4858S), phospho-S6K (Thr389, CST 9234T), phospho-4EBP1 (Thr37/46, CST 2855T), total S6 (CST 2217T), S6K (CST 9202S), 4EBP1(CST 9644T) and actin (CST 4970T) were obtained from Cell Signaling Technology.

Techniques: Injection, Control, Virus, Staining, Expressing, Labeling, Analysis

Journal: bioRxiv

Article Title: The Onset of Breathing at Birth Switches Off Regenerative Capacity

doi: 10.64898/2026.10.03.756425

Figure Lengend Snippet: a, Experimental scheme of newborns delivered under elevated 10% CO 2 conditions for in vivo imaging of AMPK-F activity. b, Representative images (left) of ExRai-AMPKAR in Vglut2-Cre pups acquired at 800 nm and 920 nm excitation wavelengths. Quantification (right) showing the emission ratio (920/800 nm excitation) of ExRai-AMPKAR. Raw ratio from the post-breathing stage was normalized to the value of the first frame to visualize the signal changes. Data are shown as mean ± SD; N = 3 biological replicates; n = 95 RGCs. ***p < 0.001, ****p < 0.0001; mixed-effects analysis with FDR correction. c, Experimental scheme of newborns delivered under reduced 10% O₂ conditions for in vivo imaging of AMPK-F activity. d, Representative images (left) of ExRai-AMPKAR in Vglut2-Cre pups acquired at 800 nm and 920 nm excitation wavelengths. Quantification (right) showing the emission ratio (920/800 nm excitation) of ExRai-AMPKAR. Raw ratio from the post-breathing stage was normalized to the value of the first frame to visualize the signal changes. Data are shown as mean ± SD; N = 5 biological replicates; n = 88 RGCs; n.s., not significant; mixed-effects analysis with FDR correction. e, Representative images of phosphorylated S6 (p-S6, green), RBPMS (red), and DAPI (blue) staining in retinal sections from 5 min postnatal pups delivered in 10% CO 2 (upper row) and 10% O 2 (bottom row). Scale bars, 200 μm (whole retina) and 50 μm (magnified views). f, Quantification showing that postnatal exposure to 10% CO 2 , but not 10% O 2 , prevents the reduction in p-S6-positive RGCs in newborns 5 min after breathing onset. Data are presented as the mean percentages of p-S6-positive cells among total RGCs in the ganglion cell layer (GCL) of each retina. ****p < 0.0001; n.s., not significant; N = 5 biological replicates for the pre-birth group and N = 5, 4 and 4 biological replicates for pups born in air, 10% CO₂ and 10% O₂ for 5 min, respectively; unpaired Welch’s t-test.

Article Snippet: Primary antibodies against phospho-S6 (Ser235/236, CST 4858S), phospho-S6K (Thr389, CST 9234T), phospho-4EBP1 (Thr37/46, CST 2855T), total S6 (CST 2217T), S6K (CST 9202S), 4EBP1(CST 9644T) and actin (CST 4970T) were obtained from Cell Signaling Technology.

Techniques: In Vivo Imaging, Activity Assay, Analysis, Staining

Journal: bioRxiv

Article Title: The Onset of Breathing at Birth Switches Off Regenerative Capacity

doi: 10.64898/2026.10.03.756425

Figure Lengend Snippet: a, Experimental scheme of adult mice inhaling 10% CO 2 for in vivo imaging. b, Representative images of donor channel (upper row), acceptor channel (middle row), and calculated FRET efficiency (bottom row) of pHLemon-rev in Vglut2-Cre adult mice acquired with 820 nm excitation after CO 2 inhalation at different time points. N = 3 biological replicates; n = 44 RGCs. c, Representative images of ExRai-AMPKAR in Vglut2-Cre adult mice acquired at 800 nm and 920 nm excitation wavelengths at different time points after CO 2 inhalation. N = 3 biological replicates; n = 68 RGCs. d, Representative images of phosphorylated S6 (p-S6, green), RBPMS (red) staining in retinal sections from adult mice that were primed either in ambient air or 10% CO 2 for 48 hr. Scale bars, 50 μm. e, Quantification showing the percentage of p-S6-positive RGCs at the indicated time points after 10% CO 2 exposure. Data are presented as the mean percentages of p-S6-positive RGCs among total RGCs in the ganglion cell layer (GCL) of each retina. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; N = 3 biological replicates for each group; unpaired Welch’s t-test. f, Summary of changes in intracellular pH, AMPK-F activity, and mTOR activity. The results of pHi are from and Extended Data Fig. 19. The results of SNRK/AMPK-F are from and Extended Data Fig. 20. The mTOR results are from . g, Representative images of optic nerves showing CTB-labeled axons at the lesion site 14 days after injury in wild-type mice. Mice were maintained either in ambient air or in 5% CO₂ starting 7 days before injury and continuing through the 14-day post-injury period. Scale bar, 100 μm. h, Quantification of CTB-labeled fluorescent intensity distal to the crush site in wild-type mice that are maintained in ambient air or 5% CO 2 environment. Data are shown as mean ± SEM; N = 7 and 10 biological replicates for ambient air and 5% CO 2 conditions, respectively; ***p < 0.001; mixed-effects analysis.

Article Snippet: Primary antibodies against phospho-S6 (Ser235/236, CST 4858S), phospho-S6K (Thr389, CST 9234T), phospho-4EBP1 (Thr37/46, CST 2855T), total S6 (CST 2217T), S6K (CST 9202S), 4EBP1(CST 9644T) and actin (CST 4970T) were obtained from Cell Signaling Technology.

Techniques: In Vivo Imaging, Staining, Activity Assay, Labeling, Analysis, Förster Resonance Energy Transfer