Fig 1: GC‐derived exosomal TAGLN2 promotes angiogenesis and endothelial dysfunction through activation of the NRP1/SEMA4D/YAP axis. (A) Representative TEM images and NTA of exosomes derived from BGC‐823 cells with TAGLN2 overexpression (TAG2‐exo), knockdown (shTAG2‐exo), or respective controls (NC‐exo or shNC‐exo). Scale bar, 100 nm. (B) Western blot analysis of exosomal markers (TSG101, CD9, CD81), the negative marker Calnexin, and TAGLN2. (C) Confocal microscopy showing intracellular uptake of PKH26‐labeled exosomes (red) by EA.hy926 cells. (D) Direct biochemical evidence of exosomal TAGLN2 interaction. Co‐IP using anti‐Flag antibody on lysates from ECs co‐cultured with exosomes derived from BGC‐823 cells stably expressing 3×Flag‐TAGLN2 or EGFP (control). Blots show Flag‐TAGLN2 and co‐precipitated endogenous NRP1, SEMA4D, SP1, and c‐Jun. (E) Representative images and quantification of cell migration and tube formation in ECs treated with 5 µg/mL of NC‐exo, shNC‐exo, shTAG2‐exo, and TAG2‐exo for 24 h. (F, G) Western blot analysis of pro‐angiogenic factors, junctional proteins, and EndoMT markers in ECs following exosome treatment. (H) Representative immunofluorescence images showing the Occludin localization at cell–cell contacts in ECs following exosome treatment. (I) Western blot analysis of core Hippo pathway components in ECs treated with exosomes. (J) Western blot validation of the axis using exosomes derived from other GC cell lines (MGC‐803 and SGC‐7901), assessing levels of TAGLN2, NRP1, SEMA4D, Hippo pathway components, and junctional proteins. (K) Western blot analysis of key signaling molecules in siNC and siPlexinB1 ECs treated with PBS, NC‐exo, or TAG2‐exo. Data are presented as mean ± SD. Significance vs. PBS control: ns, not significant; * p< 0.05, ** p< 0.01, *** p< 0.001. Lines with symbols denote pairwise comparisons.
Fig 2: TAGLN2 promotes vascular dysfunction through the NRP1/SEMA4D axis. (A) RNA‐Seq transcriptomic profiling of NRP1‐overexpressing vs. mock‐treated primary HUVECs. The top 10 enriched GO terms among significant DEGs are shown. (B) Cell migration and (C) capillary‐like tube formation were assessed following the indicated genetic manipulations (TAGLN2 or SEMA4D overexpression/knockdown, with or without concomitant modulation of NRP1). (D) Expression of core axis components (NRP1, SEMA4D), angiogenesis markers (VEGFR2, vWF), MAPK pathway markers (ERK1/2, p‐ERK1/2), and apoptosis markers (Cleaved PARP1 and Caspase 3). (E) Expression of EndoMT‐related markers: endothelial markers (E‐cadherin, Tie 1/2), mesenchymal markers (N‐cadherin, α‐SMA, FSP‐1, Vimentin), and EMT‐transcription factors (Snail, Slug, Twist, ZEB1). (F) Expression of junctional proteins (VE‐cadherin, Occludin, ZO‐1, Claudin 1, Claudin 5). (G) Immunofluorescence staining for Occludin, VE‐cadherin, and F‐actin (Phalloidin) to visualize junctional and cytoskeletal organization. (B–G) The EA.hy926 cell line was used in all experiments. Data are presented as mean ± SD. Significance vs. siNC control: ns, not significant; * p< 0.05, ** p< 0.01, *** p< 0.001. Lines with symbols denote pairwise comparisons.
Fig 3: TAGLN2 upregulates NRP1 transcription and activates YAP through the NRP1/SEMA4D axis. (A) ChIP‐qPCR analysis of endogenous TAGLN2 binding to the NRP1 promoter region. (B) Luciferase reporter assays evaluating the transcriptional activity of the NRP1 promoter (−823/+79) in response to C/EBPβ, SP1, and c‐Jun, with or without TAGLN2 overexpression. (C) Protein levels of TAGLN2, NRP1, and SEMA4D following SP1 or c‐Jun knockdown. (D) Co‐IP assays assessing endogenous interactions between TAGLN2 and SP1 or c‐Jun. (E) Levels of core Hippo pathway kinases following genetic perturbation of the TAGLN2/NRP1/SEMA4D axis. (F) Treatment with the LATS1/2 inhibitor GA‐017 (10 µM, 3 h) reversed Hippo signaling activation and junctional protein upregulation induced by knockdown of TAGLN2, NRP1, or SEMA4D. (G) Transcriptional activity of TEAD (8× GTIIC‐Luc reporter), and β‐catenin/TCF (TOPFlash/FOPFlash reporter system) following axis modulation. (H, I) Effects of SP1 or c‐Jun knockdown on Hippo pathway activity, junctional protein expression, and transcriptional activity of TEAD or β‐catenin/TCF reporters. The EA.hy926 cell line was used in all experiments. Data are presented as mean ± SD. Significance vs. respective control: ns, not significant; * p< 0.05, ** p< 0.01, *** p< 0.001. Lines with symbols denote pairwise comparisons.
Fig 4: TAGLN2 stabilizes the NRP1‐SEMA4D complex to activate YAP independently of the canonical PlexinB1‐RhoA/ROCK pathway. (A) Endogenous interaction between TAGLN2 and NRP1 was confirmed by Co‐IP using antibodies against NRP1 or the Flag tag (for exogenously expressed Flag‐NRP1). (B, C) In vitro pull‐down assays with purified recombinant proteins. Increasing amounts of TAGLN2 (100–200 µg) enhanced the direct interaction between NRP1 and SEMA4D. (D) Cycloheximide (CHX, 25 µg/mL) chase assay evaluating the protein stability of NRP1 and SEMA4D with or without TAGLN2 knockdown. (E) Subcellular fractionation and western blot analysis of TAGLN2, NRP1, SEMA4D, and YAP in EA.hy926 cells with or without TAGLN2 overexpression. (F) Competitive binding assay showing SEMA4D disrupts the cytoplasmic NRP1‐YAP interaction by Co‐IP. (G) Immunofluorescence analysis of subcellular localization for NRP1 (with or without 0.3 % Triton X‐100 pre‐permeabilization), SEMA4D, and YAP following axis modulation. Scale bar: 10 µm. (H) Quantification of capillary tube formation in EA.hy926 cells overexpressing TAGLN2, NRP1, or SEMA4D, with or without PlexinB1 knockdown. (I) Western blot analysis of key signaling molecules involved in YAP activation, ROCK suppression (p‐MYPT1, p‐MLC), and junction disassembly in EA.hy926 cells overexpressing TAGLN2, NRP1, or SEMA4D, with or without PlexinB1 knockdown. (J) Western blot analysis of indicated signaling molecules in EA.hy926 cells treated with the ROCK inhibitor Y‐27632 (10 µM, 4 h), with or without axis activation. Data are presented as mean ± SD. Significance vs. NC control: ns, not significant; * p< 0.05, ** p< 0.01, *** p< 0.001. Lines with symbols denote pairwise comparisons.
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