Journal: Biomedicines

Article Title: ATG5-FOXA3 Axis Contributes to Lysosomal Biogenesis and Auditory Function in Kölliker’s Organ

doi: 10.3390/biomedicines14040802

Figure Lengend Snippet: Conditional Atg5 deletion in SOX2 + progenitors cause moderate-to-severe sensorineural hearing loss in 2-month-old mice. ( A ) ABR audiograms of 2-month-old mice. ( B ) Representative ABR waveforms were acquired using click stimuli to display characteristic Wave I–V morphology. ( C – H ) ABR thresholds at 8–32 kHz and click show a 25- 40 dB elevation in 2-month-old Atg5flox/flox ; Sox2Cre+ mice (Red) versus controls ( Atg5flox/flox ; Sox2Cre− mice, blue). Data are mean ± SD, two-way ANOVA with Bonferroni correction. (* p < 0.05, *** p < 0.001, n = 6).

Article Snippet: Atg5flox mice (Cat#1277186, Shanghai Biomodel Organism, Shanghai, China) were crossed with Sox2- creER mice (Cat#017593, The Jackson Laboratory, Bar Harbor, ME, USA) to cochlea-supporting cell knockout.

Techniques:

Journal: Biomedicines

Article Title: ATG5-FOXA3 Axis Contributes to Lysosomal Biogenesis and Auditory Function in Kölliker’s Organ

doi: 10.3390/biomedicines14040802

Figure Lengend Snippet: FOXA3 expression mirrors the autophagy time-course and becomes progressively restricted to KO-supporting cells. ( A ) Immunofluorescence showing spatiotemporal expression of FOXA3 in the developing cochlea; FOXA3 is enriched in SOX2 + KO-cell domains, green: FOXA3; red: Myosin7a (hair-cell marker); blue: DAPI (nuclei). ( B ) Representative images illustrating that Atg5 -KD markedly reduces FOXA3 levels in KO cells, green: FOXA3; red: Myosin7a (hair-cell marker); blue: DAPI (nuclei). ( C ) Quantitative analysis of FOXA3 fluorescence intensity expression declines progressively after birth. ( D ) Quantification confirms that Atg5 -KD significantly lowers FOXA3 fluorescence intensity in KO cells. Datas are mean ± SD. (*** p < 0.01, n = 3 cochlea samples). Scale bar: 25 µm.

Article Snippet: Atg5flox mice (Cat#1277186, Shanghai Biomodel Organism, Shanghai, China) were crossed with Sox2- creER mice (Cat#017593, The Jackson Laboratory, Bar Harbor, ME, USA) to cochlea-supporting cell knockout.

Techniques: Expressing, Immunofluorescence, Marker, Fluorescence

Journal: Nature Communications

Article Title: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

doi: 10.1038/s41467-025-63053-6

Figure Lengend Snippet: a Schematic of early postnatal murine cochlear sensory epithelium. SC-subtypes (He, Hensen cells; DC1-3 Deiter’s cells 1–3, OP, outer pillar cell; IP, inner pillar cell; IPh, inner phalangeal cells) highly express SOX2 (bright green nuclei). KCs express lower levels of SOX2 (faint green nuclei). Inner HC (IHC) and three outer HCs (OHC) are labeled red. b Experimental scheme ( c – j ). Cochlear epithelial cells from stage P2 mice were used to establish control and Jag1 cKO organoid cultures, which were analyzed on day 7 of expansion. c Representative confocal images of control and Jag1 cKO organoids stained for JAG1 protein (red). Hoechst labels nuclei (blue). Scale bars = 50 µm. d RT-qPCR of relative Jag1 mRNA expression in control (Ctrl, blue bar) and Jag1 cKO (red bar) organoids ( n = 3 animals per group, three independent experiments). e Bright field (BF) images of control and Jag1 cKO organoids. Scale bars = 200 µm. f , g Organoid forming efficiency ( f ) and organoid diameter ( g ) for ( e ) ( n = 8 animals per group, three independent experiments). h Cell proliferation in control and Jag1 cKO organoids on day 7. Hoechst labels cell nuclei (blue). Scale bars = 50 µm. i Percentage of EdU + cells in ( h ) ( n = 6 animals per group, three independent experiments). j RT-qPCR of Notch target Hes1 , SC markers ( Fgf20 , Sox2, S100a1, Lfng ) and HC markers ( Atoh1 , Pou4f3 , Gfi1 , Myo7a ) in control and Jag1 cKO organoids ( n = 3 animals per group). k Experimental scheme ( l , m ). FACS-purified p27-GFP (+) SCs and p27-GFP (−) KCs from stage P2 mice were used to establish control and Jag1 cKO organoid cultures, which were analyzed on day 7 of expansion. l BF images of control and Jag1 cKO organoid cultures established from GFP (-) KCs and GFP (+) SCs. Scale bars = 200 µm. m , n Colony (organoid) forming efficiency ( m ) and organoid diameter ( n ) for ( e ) ( n = 3 independent biological replicates per group, two independent experiments). Graphed are individual data points and mean ± standard deviation. A two-tailed, unpaired Student’s t test was used to calculate exact p- values. Source data are provided as a Source Data file. N.s. not significant. Created in BioRender. Doetzlhofer, A. (2025) https://BioRender.com/vo09ioe .

Article Snippet: Sox2 CreER/+ (no. 17593), R26 rtTA*M2 (no. 006965), R26 LSLtdTomato/+ (Ai14; no. 007914), TetO-Cre tg (no. 006234), Notch1 floxed (f) (no. 007181), Notch2 floxed (no. 010525) and Notch3 -/+ (no. 010547) and Pou4f3 DTR/+ (no. 028673) mice were purchased from the Jackson Laboratories (Bar Harbor, ME).

Techniques: Labeling, Control, Staining, Quantitative RT-PCR, Expressing, Purification, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

doi: 10.1038/s41467-025-63053-6

Figure Lengend Snippet: a Experimental scheme ( b – d ). Cochlear epithelial cells from stage P2 mice were used to establish control and Jag1 cKO organoid cultures. HC formation was analyzed using RT-qPCR and immuno-staining after 5 days of differentiation. b RT-qPCR was used to analyze relative mRNA expression of early HC genes ( Atoh1 , Pou4f3 , Myo7a ) in control and Jag1 cKO organoids ( n = 3 animals per group). c Confocal images of MYO7A (magenta) and SOX2 (green) stained control and Jag1 cKO organoids. Note MYO7A and SOX2 co-staining labels new HCs. Control organoids only contain a few scattered new HCs, whereas Jag1 cKO organoids contain clusters of new HCs. Scale bars = 50 µm. d Quantification of the percentage of HCs per control and Jag1 cKO organoid in ( c ) ( n = 3 animals per group, two independent experiments). e Experimental scheme ( f – i ). FACS-purified P27-GFP (+) SCs and p27-GFP (−) KCs from stage P2 mice were used to establish control and Jag1 cKO organoid cultures. HC formation was analyzed by RT-qPCR and immuno-staining after 5 days of differentiation. f RT-qPCR was used to analyze relative mRNA expression of early HC-specific transcription factors ( Atoh1 , Gfi1 , Pou4f3 ) in control and Jag1 cKO organoids ( n = 3 independent biological replicates per group, two independent experiments). g Confocal images of KC-derived (GFP (−) ) and SC-derived (GFP (+) ) control and Jag1 cKO organoids stained for MYO7A (magenta) and SOX2 (green) Scale bars = 100 µm. h Percentage of HC-containing organoids in control and Jag1 cKO organoid cultures ( n = 4-6 independent biological replicates per group, two independent experiments). i Percentage of HCs in HC-containing organoids in control and Jag1 cKO organoid cultures ( n = 12 independent biological replicates for control and n = 8 independent biological replicates for Jag1 cKO groups). Graphed are individual data points and mean ± standard deviation. A two-tailed, unpaired Student’s t test was used to calculate exact p -values. Source data are provided as a Source Data file. N.s. not significant. Created in BioRender. Doetzlhofer, A. (2025) https://BioRender.com/vo09ioe .

Article Snippet: Sox2 CreER/+ (no. 17593), R26 rtTA*M2 (no. 006965), R26 LSLtdTomato/+ (Ai14; no. 007914), TetO-Cre tg (no. 006234), Notch1 floxed (f) (no. 007181), Notch2 floxed (no. 010525) and Notch3 -/+ (no. 010547) and Pou4f3 DTR/+ (no. 028673) mice were purchased from the Jackson Laboratories (Bar Harbor, ME).

Techniques: Control, Quantitative RT-PCR, Immunostaining, Expressing, Staining, Purification, Derivative Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

doi: 10.1038/s41467-025-63053-6

Figure Lengend Snippet: a Experimental scheme for isolating Lfng-GFP (+) cochlear SCs used in ( b ). b RT-qPCR analysis of Jag1 mRNA expression in stage P1, P5, and P13 FACS-purified cochlear SCs ( n = 2 for P1, n = 3 for P5 and n = 2 for P13. N = independent biological replicates that were obtained from at least 2 independent experiments). c Experimental scheme for ( d – g ). Cochlear explants from P2 or P5 Atoh1-nGFP tg mice were cultured with JAG1-Fc peptide or without JAG1-Fc peptide (control) in the presence of CHIR99021 and LY411575 for 4 days. EdU was added on day 1. d Representative confocal images of the HC layer taken at the cochlear mid-apical turn following culture. Newly formed HCs (white asterisks) are labeled by MYO7A (magenta) and SOX2 (blue) immuno-staining and Atoh1-nGFP (green) expression. EdU (red) labels actively and previously dividing cells. Scale bars = 25 µm. e – g Quantification of SC proliferation (EdU + SOX2 + ) ( e ), mitotic HC formation (EdU + MYO7A + ) ( f ), and the total number of newly formed HCs (MYO7A + SOX2 + Atoh1-nGFP + ) ( g ) for ( d ) ( n = 5 animals per group, two independent experiments). h Experimental scheme for ( i , j ). Cochlear explants from stage P4 Atoh1-nGFP tg mice were cultured with JAG1-Fc peptide or without JAG1-Fc peptide (control) in the presence of rapamycin (4 ng/mL) or DMSO (0.05%, vehicle control) for 5 days. CHIR99021 and LY411575 were added on day 1 to induce SC-to-HC conversion. i Representative confocal images of the HC layer taken at the cochlear mid-apical turn following culture. Newly formed HCs are marked by MYO7A (magenta) and SOX2 (blue) immuno-staining and Atoh1-nGFP (green) expression. Scale bars = 25 µm. j Quantification of newly formed HCs in ( i ) ( n = 4 animals per group, two independent experiments). Graphed are individual data points and mean ± standard deviation. Two-way ANOVA with Tukey’s correction was used to calculate exact p -values. Source data are provided as a Source Data file. N.s. not significant.

Article Snippet: Sox2 CreER/+ (no. 17593), R26 rtTA*M2 (no. 006965), R26 LSLtdTomato/+ (Ai14; no. 007914), TetO-Cre tg (no. 006234), Notch1 floxed (f) (no. 007181), Notch2 floxed (no. 010525) and Notch3 -/+ (no. 010547) and Pou4f3 DTR/+ (no. 028673) mice were purchased from the Jackson Laboratories (Bar Harbor, ME).

Techniques: Quantitative RT-PCR, Expressing, Purification, Cell Culture, Control, Labeling, Immunostaining, Standard Deviation

Journal: Nature Communications

Article Title: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

doi: 10.1038/s41467-025-63053-6

Figure Lengend Snippet: a Representative confocal image of JAG1 expression in undamaged cochlear sensory epithelia from stage P3 wild-type mice, and in HC-damaged cochlear sensory epithelia from stage P3 Pou4f3 DTR/+ transgenic mice that received diphtheria toxin (DT) at P1 (two independent experiments). Scale bars=50 µm. b Experimental scheme for ( c - f ). Cochlear explants from stage P5 wild-type mice were cultured with gentamicin to ablate HCs, followed by culturing them with JAG1-Fc peptide or without JAG1-Fc peptide (control) in the presence of CHIR99021 and LY411575 . EdU was added to the culture media on day two to analyze cell proliferation. c Representative confocal images of the HC layer taken at the cochlear mid-apex and base following culture. Newly formed HCs are marked by MYO7A (magenta) and SOX2 (blue) immuno-staining, indicated by yellow asterisks. EdU (green) labels actively dividing SOX2 + SCs (white asterisks) and previously dividing MYO7A + SOX2 + HCs (red asterisks). Scale bars=25 µm. d-f Quantification of EdU + SOX2 + cells in HC layer (HCs and SCs) (d), new HCs (MYO7A + SOX2 + ) in mid-apex ( e ) and new HCs in base ( f ) for c ( n = 4 animals per group, two independent experiments). Graphed are individual data points and mean ± standard deviation. Two-tailed, unpaired Student’s t test was used to calculate exact p -values. Source data are provided as a Source Data file. N.s. not significant.

Article Snippet: Sox2 CreER/+ (no. 17593), R26 rtTA*M2 (no. 006965), R26 LSLtdTomato/+ (Ai14; no. 007914), TetO-Cre tg (no. 006234), Notch1 floxed (f) (no. 007181), Notch2 floxed (no. 010525) and Notch3 -/+ (no. 010547) and Pou4f3 DTR/+ (no. 028673) mice were purchased from the Jackson Laboratories (Bar Harbor, ME).

Techniques: Expressing, Transgenic Assay, Cell Culture, Control, Immunostaining, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

doi: 10.1038/s41467-025-63053-6

Figure Lengend Snippet: a Experimental scheme. AAV expressing ATOH1 and EGFP (or EGFP only) was injected with or without JAG1-Fc peptide into the cochlea of P7 wild-type mice through the round window membrane. 7 days later, injected cochleae were immuno-stained for SOX2 (blue) and MYO7A (magenta) to identify newly formed HCs (MYO7A + SOX2 + ). EGFP (green) marks infected cells. b Representative confocal images of the HC layer at the cochlear apex and base that received JAG1-Fc peptide (JAG1-Fc peptide + AAV-EGFP) or ATOH1 expressing AAV (PBS + AAV-ATOH1-EGFP) (control) or both JAG1-Fc peptide and ATOH1 expressing AAV (JAG1-FC peptide + AAV-ATOH1-EGFP). Scale bar=25 μm. c Quantification of infected cells that formed HCs (SOX2 + MYO7A + ) in the cochlear apex and base in ( b ) (graphed are mean ± SD, n = 6 mice per group, three independent experiments). One-way ANOVA with Tukey’s correction was used to calculate exact p -values. Source data are provided as a Source Data file. N.s. not significant. Created in BioRender. Doetzlhofer, A. (2025) https://BioRender.com/m18j826 .

Article Snippet: Sox2 CreER/+ (no. 17593), R26 rtTA*M2 (no. 006965), R26 LSLtdTomato/+ (Ai14; no. 007914), TetO-Cre tg (no. 006234), Notch1 floxed (f) (no. 007181), Notch2 floxed (no. 010525) and Notch3 -/+ (no. 010547) and Pou4f3 DTR/+ (no. 028673) mice were purchased from the Jackson Laboratories (Bar Harbor, ME).

Techniques: Expressing, Injection, Membrane, Staining, Infection, Control

Journal: Scientific Reports

Article Title: Neurogenesis from Sox2 expressing cells in the adult cerebellar cortex

doi: 10.1038/s41598-017-06150-x

Figure Lengend Snippet: Undifferentiated Sox2 + cells express stemness marker Nestin and show reactive properties. Undifferentiated Sox2 + cells are characterized by Nestin expression, detected by Nestin-dependent GFP expression and the lack of Blbp expression ( a–c ). Additionally, Sox2 + Nestin + cells show proliferative capacity as shown by Ki67 labeling ( d–f ). Sox2 + S100 − cells were sparsely distributed in the PCL of control animals ( g–i ) when compared to animals that received a week of physical training (arrows in j–l ). Training of mice on the RotaRod system lead to a significant increase in the number of Sox2 + S100 − cells within the PCL as compared to controls ( m – Mann-Whitney-U test; SEM). Immunohistochemical staining in panels a – f was performed on 4 µm FFPE sections. Staining shown in panels g – l was performed on 50 µm free-floating vibratome sections. White lines show perimeter of Purkinje cell layer. Scale bars equate to 50 µm in panels a – f and 100 µm in panels g – l.

Article Snippet: Adult Sox2-creER T2 ::tdTomato -mice (>60 days postpartum ) were either trained on the RotaRod system (RotaRod advanced; TSE Systems, Bad Homburg, Germany) or placed in an enriched environment (EE) to study reactivity of progenitors and neurogenesis.

Techniques: Marker, Expressing, Labeling, Control, MANN-WHITNEY, Immunohistochemical staining, Staining