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: To measure surface populations of SDC4, resuspended cells (>10,000) were incubated with FITC-conjugated anti-SDC4 or its isotype-matched control antibody (sc-12766, Santa Cruz) for 15 min on ice and processed for flow cytometry analysis following the manufacturer’s instructions.

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

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: Proximity ligation assay was performed according to the protocol provided by Sigma-Aldrich (DUO092101), and the primary antibodies and dilutions are anti-SDC4 (sc12766, 1:100), anti-Syntenin (22399-1-AP, 1:400, Proteintech).

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

Journal: bioRxiv

Article Title: IP6K1 interacts with the syndecan SDC4 to support secretory granule biogenesis in gastric chief cells

doi: 10.1101/2025.09.17.676719

Figure Lengend Snippet: (A) AGS cells were transfected to express either SFB-tagged IP6K1 or SFB-GFP as a control. SFB-tagged proteins were purified by tandem-affinity purification using streptavidin beads followed by S-protein beads in two replicates for each set. The Venn diagram illustrates the total number of interacting proteins identified across both replicates for each condition, with the numbers of GFP- and IP6K1-binding proteins indicated above. B) The Venn diagram illustrates IP6K1 interacting proteins identified earlier in HEK293T cells , in comparison with IP6K1 interactors in AGS cells identified in this study. C) GST-IP6K1 or GST alone were transiently overexpressed in wild-type AGS cells with SDC4-mCherry, and pulled down on glutathione agarose beads. The GST tag was detected using an anti-GST antibody to confirm the expression and successful pull-down of GST-tagged proteins. The experimental replicate data (1 and 2) shown here are representative of three independent experiments (N=3). D) Representative immunoblots show the co-immunoprecipitation of overexpressed SDC4-mCherry with myc-tagged IP6K paralogues. AGS cells were transiently transfected with plasmids encoding myc-IP6K1, myc-IP6K2, or myc-IP6K3. IP6Ks were immunoprecipitated and probed for SDC4-mCherry using an anti-mCherry rabbit polyclonal antibody. myc-tagged IP6Ks were detected using an anti-myc mouse monoclonal antibody (N=3). Non-specific bands in the input sample are marked with an asterisk (*). E) The image shows triple staining of fixed AGS cells for IP6K1-V5 (orange), SDC4-GFP (green), and PGC-mCherry (magenta). The corresponding intensity profile confirms partial co-localisation of these three proteins within the cell. F) Co-expression of SDC4-GFP (green) with PGC-mCherry (magenta) in IP6K1 +/+ and IP6K1 -/- AGS cells. Dashed white lines mark the cell boundaries Scale bar 10 µm. G) Zoomed in images of the region in (F) marked by yellow boxes. Scale bar 2 μm. The traces display the fluorescence intensity profile for SDC4 (green) and PGC (magenta), measured along the red arrow in the figure. H) Live cell imaging was used to evaluate the co-migration of SDC4 and PGC in IP6K1 +/+ cells compared to IP6K1 -/- AGS cells. Still frames captured within 60 seconds indicate co-migration of SDC4 (green) and PGC (red) granules, marked by white arrowheads in IP6K1 +/+ cells. Scale bar: 1 μm. Super-resolution images in (E, F,G and H) were obtained with the Elyra 7 (SIM) module on a Zeiss LSM 980 confocal microscope, using a 63×/1.4 NA objective.

Article Snippet: Primary antibodies used for immunoblotting (IB), immunofluorescence (IF), immunoprecipitation (IP), along with their dilutions, were as follows: anti-IP6K1 (Santa Cruz Biotechnology, sc-376290; 1:1000 IB; Sigma, HPA040825; 1:400 IF); anti-pepsinogen C (Abcam, ab180709; 1:5000 IB; 1:2000 IF); anti Lipase F (Sigma, HPA045930; 1:5000 IB;1:500 IF); anti E-cadherin (CST, 14472S; 1:500 IF); anti α-tubulin (Sigma-Aldrich, T9025; 1:5000 IB); anti β-actin (Sigma-Aldrich A2228; 1:5000 IB); anti SDC4 (Santa Cruz Biotechnology, sc-12766; 1:50 IF; 1:1000 IB); anti-GFP (Invitrogen, A11122; 1:5000 IB; 2 μg IP); anti-GST (Abcam, ab19256; 1:3000 IB), anti-cMyc (Sigma-Aldrich, M4439; 1:10,000 IB; 1 μg IP; 1:800 IF); anti mCherry (Abcam, ab183628; 2μg IP; 1:5000 IB).

Techniques: Transfection, Control, Purification, Affinity Purification, Binding Assay, Comparison, Expressing, Western Blot, Immunoprecipitation, Staining, Fluorescence, Live Cell Imaging, Migration, Microscopy

Journal: bioRxiv

Article Title: IP6K1 interacts with the syndecan SDC4 to support secretory granule biogenesis in gastric chief cells

doi: 10.1101/2025.09.17.676719

Figure Lengend Snippet: A) Immunofluorescence images of AGS cells show ANXA2 (green, top row) and SDC4 (green, bottom row) localization, with nuclei counterstained using DAPI (blue); both proteins display cytoplasmic and membrane-associated distribution (Scale bars 10 μm). B) Immunoblot analysis of SDC4 in stomach tissues of Ip6k1 +/+ mice and Ip6k1 −/− mice. β-actin was used as a loading control. The asterisk (*) indicates the specific band corresponding to SDC4 in the stomach tissues. SDC4 expression in stomach tissue was examined using the same lysates as shown in for comparison. C) Quantification of SDC4 expression in adult Ip6k1 +/+ and Ip6k1 −/− mouse stomach. Values indicate mean ± SEM; N=3 for each genotype. Data were analysed using a two-tailed Student’s t-test (ns non-significant; P ≥ 0.05). D) AGS cell extracts were immunoprecipitated using an antibody against the N-terminus of IP6K1, and normal rabbit IgG was used as a control. The presence of SDC4 was detected using an anti-SDC4 monoclonal antibody. β-actin was used as a loading control for input samples (N=3).

Article Snippet: Primary antibodies used for immunoblotting (IB), immunofluorescence (IF), immunoprecipitation (IP), along with their dilutions, were as follows: anti-IP6K1 (Santa Cruz Biotechnology, sc-376290; 1:1000 IB; Sigma, HPA040825; 1:400 IF); anti-pepsinogen C (Abcam, ab180709; 1:5000 IB; 1:2000 IF); anti Lipase F (Sigma, HPA045930; 1:5000 IB;1:500 IF); anti E-cadherin (CST, 14472S; 1:500 IF); anti α-tubulin (Sigma-Aldrich, T9025; 1:5000 IB); anti β-actin (Sigma-Aldrich A2228; 1:5000 IB); anti SDC4 (Santa Cruz Biotechnology, sc-12766; 1:50 IF; 1:1000 IB); anti-GFP (Invitrogen, A11122; 1:5000 IB; 2 μg IP); anti-GST (Abcam, ab19256; 1:3000 IB), anti-cMyc (Sigma-Aldrich, M4439; 1:10,000 IB; 1 μg IP; 1:800 IF); anti mCherry (Abcam, ab183628; 2μg IP; 1:5000 IB).

Techniques: Immunofluorescence, Membrane, Western Blot, Control, Expressing, Comparison, Two Tailed Test, Immunoprecipitation

Journal: bioRxiv

Article Title: IP6K1 interacts with the syndecan SDC4 to support secretory granule biogenesis in gastric chief cells

doi: 10.1101/2025.09.17.676719

Figure Lengend Snippet: Illustration highlights the role of inositol phosphate kinase 1 (IP6K1) in regulating gastric digestion. Deletion of IP6K1 reduces serum protein and increases faecal protein, correlating with muscle loss and reduced body weight. Different cell types are marked in a representation of a gastric gland. The model highlights IP6K1’s role in gastric digestion through PGC and LIPF granule formation and secretion in chief cells. Finally, it shows that IP6K1 acts via SDC4 to facilitate PGC granule trafficking and release.

Article Snippet: Primary antibodies used for immunoblotting (IB), immunofluorescence (IF), immunoprecipitation (IP), along with their dilutions, were as follows: anti-IP6K1 (Santa Cruz Biotechnology, sc-376290; 1:1000 IB; Sigma, HPA040825; 1:400 IF); anti-pepsinogen C (Abcam, ab180709; 1:5000 IB; 1:2000 IF); anti Lipase F (Sigma, HPA045930; 1:5000 IB;1:500 IF); anti E-cadherin (CST, 14472S; 1:500 IF); anti α-tubulin (Sigma-Aldrich, T9025; 1:5000 IB); anti β-actin (Sigma-Aldrich A2228; 1:5000 IB); anti SDC4 (Santa Cruz Biotechnology, sc-12766; 1:50 IF; 1:1000 IB); anti-GFP (Invitrogen, A11122; 1:5000 IB; 2 μg IP); anti-GST (Abcam, ab19256; 1:3000 IB), anti-cMyc (Sigma-Aldrich, M4439; 1:10,000 IB; 1 μg IP; 1:800 IF); anti mCherry (Abcam, ab183628; 2μg IP; 1:5000 IB).

Techniques: