Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: MG132 inhibits the degradation of SDC4-CTF. A and B , Western blot of HCT116 cells treated with 0.5, 1, 2.5, or 5 μM MG132 for 12 h, or with 2.5 μM MG132 for 0 to 12 h. C , Western blot of full-length SDC1 and SDC4 in HCT116 cells treated with MG132 (0.5–5 μM, 12 h). D and E , immunofluorescence detection and quantification of HCT116 cells transfected with SDC1-GFP or SDC4-GFP, with/without 5 μM MG132 (4 h; n = 6). F , Western blot of multiple colorectal cancer cell lines treated with 10 μM MG132 (12 h). G , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with 0.1, 0.5, or 1 μM proteasome inhibitors (Carfilzomib, Ixazomib, Bortezomib) for 24 h. H , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with PD150606 (2, 5, 10 μM) for 12 h. I , quantification of DQ-BSA fluorescence with MG132 or Earle’s balanced salt solution (EBSS) treatment (n = 4). J , quantification of lysosomal activity using LysoTracker with MG132 or EBSS treatment (n = 4). K , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells with siRNA knockdown of proteasome subunits PSMD14, PSMD2, USP14, PSMB5, PSMA6. Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.0001). SDC4, syndecan4; CTF, C-terminal transmembrane (TM) fragment; EBSS, Earle’s balanced salt solution.

Article Snippet: Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam.

Techniques: Western Blot, Immunofluorescence, Transfection, Fluorescence, Activity Assay, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: SDC4-CTF is further cleaved by γ-secretase to release the cytoplasmic fragment. A , confocal microscopy of HCT116 cells transfected with SDC4-GFP or SDC4-CTF-GFP. B , schematic of SDC4-GFP deletion mutants, SNP for Signal peptide, ED for ectodomain, TM for TM motif, C1 and C2 for the constant regions, and V for variable region. C , Western blot of HCT116 cells transfected with SDC1-GFP, SDC4-GFP, or SDC4-CTF-GFP, with/without 5 μM MG132 (4 h). D , sequence alignment of Syndecan family TM C-terminal fragments. The alignment was performed using Clustal W, and analysis was done with ESPript 3.0. E , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells with presenilin-1 knockdown (siRNA, 72 h). F , Western blot of HCT116 and SW480 cells treated with 10 μM DAPT, 10 μM MG132, or 2.5 μM GM6001 (12 h). G , schematic of SDC4-CTF degradation via lysosome or proteasome pathways. Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗ p < 0.001, ∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.0001). SDC4, Syndecan4; CTF, C-terminal transmembrane fragment.

Article Snippet: Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam.

Techniques: Confocal Microscopy, Transfection, Western Blot, Sequencing, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: Syntenin stabilizes SDC4-CTF against endocytic-lysosomal degradation. A , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with 25 μM LY294002, 250 nM Rapamycin, or 250 nM Bafilomycin A1. B , Western blot of HCT116 cells starved in EBSS for indicated durations. C and D , Western blot and quantification of SDC1-CTF and SDC4-CTF in HCT116 cells overexpressing Flag-tagged Syntenin (n = 3). E , schematic and sequences of SDC4 WT and SDC4 ΔC2 , with confocal images of indicated plasmids in 293T cells. F , quantification of fluorescence co-localization from ( E ) (n = 6). G and H , Western blot and quantification of SDC4-CTF and Syntenin in 293T cells co-transfected with SDC4 and GFP-tagged Syntenin (n = 3). I , Western blot of SDC1-CTF, SDC4-CTF, and Syntenin in multiple cancer cell lines. J , correlation analysis between SDC4-CTF and Syntenin expression from ( I ). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗ p < 0.001, ∗∗∗ p < 0.0005, ns). SDC4, Syndecan4; CTF, C-terminal transmembrane fragment; EBSS, Earle’s balanced salt solution.

Article Snippet: Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam.

Techniques: Western Blot, Fluorescence, Transfection, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: AGO binds to SDC4 and disrupts SDC4-Syntenin interaction. A , chemical structure of AGO. B , microscale thermophoresis (MST) assays of AGO with GFP-SDC4 or GFP control in 293T lysate (n = 3). C , MST assays of AGO with recombinant glutathione S-transferase (GST)-SDC4 (n = 3). D and E , MST assays of AGO with GFP-tagged SDC4 or SDC1 and their TM deletion mutants (n = 3 each). F and G , Western blot and quantification of AGO drug affinity responsive target stability experiments (n = 3). H and I , immunoprecipitation (IP) and quantification of SDC4 binding to Syntenin in SW480 cells treated with 20 μM AGO (24 h; n = 3). J , IP of Flag-Syntenin and GFP-SDC4 in 293T cells treated with 20 μM AGO (24 h). K , GST pull-down of Syntenin from 293T lysate with/without 20 μM AGO, using GST or GST-SDC4. L and M , poximity ligation assays and quantification of SDC4-Syntenin interaction in SW480 cells treated with 20 μM AGO (24 h; n = 8). O and P , confocal images and quantification of SDC4/GFP-Syntenin co-localization in 293T cells treated with 20 μM AGO (24 h; n = 8). Q , IP of SDC4 and detection of GFP-Syntenin/endogenous Syntenin in 293T cells treated with 20 μM AGO (24 h). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗ p < 0.001, ∗∗∗ p < 0.0005, ns). AGO, Andrographolide; GST, glutathione S-transferase; SDC4, Syndecan4; MST, microscale thermophoresis; TM, transmembrane; IP, immunoprecipitation.

Article Snippet: Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam.

Techniques: Microscale Thermophoresis, Control, Recombinant, Western Blot, Immunoprecipitation, Binding Assay, Ligation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: AGO promotes lysosomal degradation of SDC4-CTF. A , Western blot of SDC1/4 CTF and full-length in SW480 cells treated with AGO (indicated doses, 24 h; n = 3). B , Western blot in SW480 cells treated with 20 μM AGO for 0 to 24 h. C and D , immunofluorescence detection and quantification of SDC4 in SW480 cells treated with 20 μM AGO (24 h; n = 6). E , surface SDC4 measured by FACS using FITC-anti-SDC4. F , Western blot of SDC1/4 CTF and full-length in HCT116 cells treated with AGO (indicated doses, 24 h). G , Western blot of SDC4-CTF in multiple colorectal cancer cells treated with 20 μM AGO (24 h). H , Western blot of SDC1-CTF and SDC4-CTF in SW480 cells treated with cycloheximide (50 μg/ml) along with/without 20 μM AGO for indicated times. I , RT-qPCR of indicated genes in SW480 cells treated with 20 μM AGO (24 h; n = 4). J , Western blot of SDC1-CTF and SDC4-CTF in SW480 cells treated with 20 μM AGO, 2.5 μM GM6001, 10 μM TMI-1, or combinations (24 h). K , Western blot in SW480 cells treated with 20 μM AGO, 5 μM BAY11 to 7082, 10 nM PMA, 1 μg/ml LPS, or 20 μg/ml TNFα (24 h). L and M , live-cell imaging and analysis of SDC4-GFP intensity in 293T cells co-expressing DsRed-Rab5A and SDC4-GFP, with 20 μM AGO treatment (n = 4). O , Western blot of lysosome markers and SDC1/4-CTF in SW480 cells treated with 20 μM AGO, 250 nM BAF1, or combination (24 h). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns). AGO, andrographolide; SDC4, Syndecan4; CTF, C-terminal transmembrane fragment; FACS, fluorescence-activated cell sorting; BAF1, Bafilomycin A1.

Article Snippet: Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam.

Techniques: Western Blot, Immunofluorescence, Quantitative RT-PCR, Live Cell Imaging, Expressing, Two Tailed Test, Fluorescence, FACS