Journal: bioRxiv

Article Title: PROX1 loss in adult mouse Schlemm’s canal causes permanent ocular hypertension

doi: 10.1101/2025.11.13.688015

Figure Lengend Snippet: Human and mouse SC endothelial cells share a lymphatic-like hybrid phenotype mediated by PROX1. a- Whole mount immunostaining and cryo cross-sections of mouse ( a ) and human ( b ) Schlemm’s canals revealed robust expression of the lymphatic markers PROX1 and FLT4. (c) PROX1 and CCL21 expression were reduced in primary human SC endothelial cells treated with PROX1 siRNA, while levels of the universally expressed endothelial gene TEK were unchanged when measured by real-time qPCR (siControl n = 6, si PROX1 n = 6). ( d , quantified in e ) Western blot revealed reduced expression of FLT4 protein in siPROX1-treated human SC cells (n = 3 per group). ( f ) Reduced expression of lymphatic genes and increased expression of blood endothelial genes were detected in si PROX1 -treated SC cells by RNA sequencing, accompanied by increased expression of TGFB-signaling genes and reduction in cell proliferation markers (n = 3 per group). ( g , quantified in h ) Confocal microscopy of eye flat mounts revealed reduced PROX1 and FLT4 expression in Prox1 flox/flox ; Cdh5 CreERT2 mice 4 weeks after tamoxifen induction at 8 weeks of age ( Prox1 ΔEC). While Prox1 deletion was generally robust, some mosaicism was observed, and a small number of PROX1-positive nuclei were observed in Prox1 ΔEC SC (white arrowheads, Prox1 ΔEC n = 3, Control n = 4). Dashed lines in g outline Schlemm’s canal. ( i ) No change in IOP ( Prox1 ΔEC n = 9, Control n = 7) or ( j ) Schlemm’s canal width ( Prox1 ΔEC n = 3, Control n = 4) was measured 4 weeks after tamoxifen induction. * p <0.05, ** p <0.01 as determined by 2-tailed unpaired Student’s t test. Error bars in c , e , h and i indicate ±SEM, while each point denotes an independent biological replicate.

Article Snippet: Primary antibodies used: Mouse anti PROX1 (Developmental Studies Hybridoma Bank, Iowa City, IA USA, 1A6), Goat anti LYVE1 (R&D systems AF2125), Goat anti FLT4 (R&D systems, AF349), Rabbit anti ERG (Abcam, Waltham MA, USA, ab92513).

Techniques: Immunostaining, Expressing, Western Blot, RNA Sequencing, Confocal Microscopy, Control

Journal: bioRxiv

Article Title: PROX1 loss in adult mouse Schlemm’s canal causes permanent ocular hypertension

doi: 10.1101/2025.11.13.688015

Figure Lengend Snippet: SC-specific Prox1 deletion leads to ocular hypertension . ( a ) Schematic of experimental timeline used for nanocarrier induction of Prox1 deletion. ( b , c ) 4 weeks after nanocarrier induction, IOP elevation was observed in eyes of Prox1 flox/flox ; Cdh5 -CreERT2 mice receiving 4OH tamoxifen (4OHT) nanocarriers in comparison to contralateral eyes receiving empty (blank) nanocarriers. IOP elevation persisted throughout the duration of the experiment. No IOP elevation was seen in Cre-negative mice treated with 4OHT nanocarriers. (Cre+, n = 8, Cre-, n = 8). ( d ) In vivo visible light-OCT imaging 12 weeks after nanocarrier-mediated Prox1 deletion revealed no change in canal size on B-scans, or ( e ) longitudinal reconstructions (pseudo-colored in yellow) of Schlemm’s canal. ( f ) Comparison of luminal area by 16 individual OCT B scans captured around the circumference of Schlemm’s canal showed no difference in canal size between matched 4OHT and blank nanocarrier-treated eyes of Cdh5 -CreERT2-positive mice. ( g ) PROX1 expression and canal morphology were examined in Schlemm’s canal flat mounts collected 6 months after nanocarrier induction by confocal microscopy. Dashed lines in PROX1 panels indicate the outline of PECAM1-positive Schlemm’s canal. ( h ) Compared with contralateral control eyes, PROX1 expression was significantly reduced in eyes treated with 4OHT nanocarriers. ( i ) No change in Schlemm’s canal size was observed in 4OHT nanocarrier-treated eyes (n = 6). * p < 0.05, ** p < 0.01, **** p < 0.0001 as determined by 2-way ANOVA followed by Bonferroni posttests ( b , c and f ) or 2-tailed paired Student’s t test ( h & i ). Error bars in b , c , h , and i indicate ±SEM, while each point represents an independent biological replicate. Each point in f represents a single measurement captured along the length of Schlemm’s canal, while each violin represents a single eye.

Article Snippet: Primary antibodies used: Mouse anti PROX1 (Developmental Studies Hybridoma Bank, Iowa City, IA USA, 1A6), Goat anti LYVE1 (R&D systems AF2125), Goat anti FLT4 (R&D systems, AF349), Rabbit anti ERG (Abcam, Waltham MA, USA, ab92513).

Techniques: Comparison, In Vivo, Imaging, Expressing, Confocal Microscopy, Control

Journal: bioRxiv

Article Title: PROX1 loss in adult mouse Schlemm’s canal causes permanent ocular hypertension

doi: 10.1101/2025.11.13.688015

Figure Lengend Snippet: ( a ) Representative toluidine blue-stained semithin cross-sections of Schlemm’s canal from Prox1 flox/flox ; Cdh5 -CreERT2 eyes 12 weeks after treatment with blank or 4OHT nanocarriers. Semithin sections were imaged by light microscopy and used to measure ( b ) SC cross sectional area, ( c ) canal height (depth) and ( d ) Feret diameter (longest axis; n = 3 pairs of eyes from Cdh5 -CreERT2-positive nanocarrier-treated animals). Schlemm’s canal lumen indicated by red shading. When compared to same-animal contralateral control eyes, no significant difference in canal size or morphology was observed. ( e ) Representative TEM images from the inferior quadrant of matching eyes treated with 4OHT or blank nanocarriers from a Prox1 flox/flox ; Cdh5 -CreERT2 mouse revealed no differences in SC inner wall or juxtacanalicular meshwork morphology 12 weeks after nanocarrier injection. Giant vacuoles (GV) were observed in the inner wall of both 4OHT-treated and contralateral control eyes. Statistical comparisons in b - d were performed using a Bonferroni-corrected 2-tailed, paired Student’s t test. Error bars in b , c , and d indicate ±SEM, while each point represents an independent biological replicate.

Article Snippet: Primary antibodies used: Mouse anti PROX1 (Developmental Studies Hybridoma Bank, Iowa City, IA USA, 1A6), Goat anti LYVE1 (R&D systems AF2125), Goat anti FLT4 (R&D systems, AF349), Rabbit anti ERG (Abcam, Waltham MA, USA, ab92513).

Techniques: Staining, Light Microscopy, Control, Injection

Journal: IBRO Neuroscience Reports

Article Title: Prospero-related homeobox protein 1 (Prox1) enhances myelin sheath regeneration in injured sciatic nerve

doi: 10.1016/j.ibneur.2025.09.011

Figure Lengend Snippet: Double IF staining of Prox1 (red) and MBP (green) with IF (×200) and their intensity profile obtained using ImageJ software, along an ideal straight line (white).

Article Snippet: Rabbit anti-Myelin basic protein/MBP Polyclonal antibody (Proteintech, USA, catalog # 10458–1-AP); Mouse anti-MBP tag Monoclonal antibody (Proteintech, USA, catalog # 66003–1-Ig); Anti-PROX1 Antibody (Boster Bio, China, catalog # A01985–1); DAB Chromogenic Substrate Reagent Kit (Blue) (Boster Bio, China, catalog # AR1025); Rabbit Anti-GAPDH (Jiangsu KeyGEN BioTECH, China, catalog # KGC6102–1); Luxol Fast Blue Myelin Stain Kit (Solarbio, China, G3240); KeygenMAX 3000 transfection reagent (Jiangsu KeyGEN BioTECH, China, catalog # KGA9705–1); PrimeScriptTM RT Master Mix (TaKaRa, Japan, catalog # RR036B); Olympus Inverted Microscope (OLYMPUS, Japan, catalog # IX51); ABI Stepone real-time PCR (Thermo Fisher, USA); Trans-Blot® TurboTM Transfer System (BIO-RAD, USA, catalog #1704150).

Techniques: Staining, Software

Journal: IBRO Neuroscience Reports

Article Title: Prospero-related homeobox protein 1 (Prox1) enhances myelin sheath regeneration in injured sciatic nerve

doi: 10.1016/j.ibneur.2025.09.011

Figure Lengend Snippet: (A) IHC images of Prox1 proteins in sciatic nerve tissue of control sciatic nerves and injured sciatic nerves on Day 3, 4, 5, 6, 7 and 14 post-injuries (×200) and the quantification of Prox1 proteins in top right panel (P value was normalized to control group, **** P < 0.0001, n = 5, One-Way ANOVA analysis was used). (B) Western blot and quantitative analysis of Prox1 protein expression in sciatic nerve tissue from control nerves and injured nerves at days 3, 4, 5, 6, 7, and 14 post-injury (**** P < 0.0001, n = 3, One-Way ANOVA analysis was used).

Article Snippet: Rabbit anti-Myelin basic protein/MBP Polyclonal antibody (Proteintech, USA, catalog # 10458–1-AP); Mouse anti-MBP tag Monoclonal antibody (Proteintech, USA, catalog # 66003–1-Ig); Anti-PROX1 Antibody (Boster Bio, China, catalog # A01985–1); DAB Chromogenic Substrate Reagent Kit (Blue) (Boster Bio, China, catalog # AR1025); Rabbit Anti-GAPDH (Jiangsu KeyGEN BioTECH, China, catalog # KGC6102–1); Luxol Fast Blue Myelin Stain Kit (Solarbio, China, G3240); KeygenMAX 3000 transfection reagent (Jiangsu KeyGEN BioTECH, China, catalog # KGA9705–1); PrimeScriptTM RT Master Mix (TaKaRa, Japan, catalog # RR036B); Olympus Inverted Microscope (OLYMPUS, Japan, catalog # IX51); ABI Stepone real-time PCR (Thermo Fisher, USA); Trans-Blot® TurboTM Transfer System (BIO-RAD, USA, catalog #1704150).

Techniques: Control, Western Blot, Expressing

Journal: IBRO Neuroscience Reports

Article Title: Prospero-related homeobox protein 1 (Prox1) enhances myelin sheath regeneration in injured sciatic nerve

doi: 10.1016/j.ibneur.2025.09.011

Figure Lengend Snippet: MBP protein level was significantly increased by PROX1 overexpression. (A) qPCR quantification of PROX1 overexpression, (B) WB images of MBP and GAPDH protein from cells transfected with NC, pcDNA-vector or pcDNA-prox1, (C) the quantification of (B). (**** P < 0.0001, n = 3, One-Way ANOVA analysis was used).

Article Snippet: Rabbit anti-Myelin basic protein/MBP Polyclonal antibody (Proteintech, USA, catalog # 10458–1-AP); Mouse anti-MBP tag Monoclonal antibody (Proteintech, USA, catalog # 66003–1-Ig); Anti-PROX1 Antibody (Boster Bio, China, catalog # A01985–1); DAB Chromogenic Substrate Reagent Kit (Blue) (Boster Bio, China, catalog # AR1025); Rabbit Anti-GAPDH (Jiangsu KeyGEN BioTECH, China, catalog # KGC6102–1); Luxol Fast Blue Myelin Stain Kit (Solarbio, China, G3240); KeygenMAX 3000 transfection reagent (Jiangsu KeyGEN BioTECH, China, catalog # KGA9705–1); PrimeScriptTM RT Master Mix (TaKaRa, Japan, catalog # RR036B); Olympus Inverted Microscope (OLYMPUS, Japan, catalog # IX51); ABI Stepone real-time PCR (Thermo Fisher, USA); Trans-Blot® TurboTM Transfer System (BIO-RAD, USA, catalog #1704150).

Techniques: Over Expression, Transfection, Plasmid Preparation