A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death
- National Cancer Institute, National Institutes of Health, United States
- APCBIo Innovations Inc., United States
- Frederick National Laboratory for Cancer Research, Leidos Biomedical Inc., United States
Figures
Figure 1
SCGB3A2-induced suppression of LLC cell proliferation.
(A) Effect of SCGB3A2 on proliferation of LLC cells. Cells were maintained without serum for 24 hr, followed by 1% FBS-RPMI1640 media with or without mouse SCGB3A2 (1 μg/ml). CCK8 assay was carried …
Figure 2 with 1 supplement
SCGB3A2 as an LPS binding protein.
(A) CCK8 analysis using various concentrations as shown in the bottom (pg, ng/ml) of smooth LPS (E.coli O111:B4 serotype) and rough LPS (Ra-LPS) after 72 hr in culture. C; control without any …
Figure 2—figure supplement 1
Analysis of LPS-SCGB3A2 complex.
(A) CCK8 analysis using various recombinant SCGB3A2s (1 µg/ml) obtained from different sources/batches. LLC cells grown in 1% FBS-RPMI 1640 medium were harvested at 72 hr and analyzed. Averages ± SD …
Figure 3 with 1 supplement
SCGB3A2 binding to LLC cells through HS of SDC1.
(A) Schematic model of a human protein array for the isolation process of candidate genes shown as a Venn diagram. (B) Co-immunoprecipitation assay of SCGB3A2-FLAG and SDC1-Myc-His in COS-1 cells. …
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Figure 3—source data 1
Results of whole protein-protein interaction array.
- https://doi.org/10.7554/eLife.37854.008
Figure 3—figure supplement 1
Cell surface expression of SDC1 and validation of sh-SDC1 and ARH-77-mSDC1 clones.
(A) Representative IHC for SDC1 in lung metastasized LLC cells in mice. Bar = 200 µm (upper), 50 µm (bottom). Bottom is magnified image of red rectangle area. (B) LLC and B16F10 cells grown in 10% …
Figure 4 with 5 supplements
SCGB3A2-LPS uptake activates inflammasome signaling.
(A) Immunofluorescence analysis of LLC and ARH-77-mSDC1 cells for SCGB3A2, SDC1, and ICAM-1. Arrowheads: uropod-like structures. Cells were incubated in 0% FBS-RPMI 1640 for 40 min (0%40 m) with or …
Figure 4—figure supplement 1
Analysis of SCGB3A2-HaloTag protein trafficking.
(A) Live cell imaging of LPSA488 binding to LLC cells. LLC cells were incubated in 1%FBS-RPMI1640 with LPSA488 (1 µg/ml) for 16 hr with HaloTag control vector-transfected supernatant (SN) or mouse …
Figure 4—figure supplement 2
SCGB3A2 modeling.
(A) Side-by side comparison of 1UTG (uteroglobin, SCGB1A1) structure (PDBID:1UTG) and our model (SCGB3A2). S4-A-2 shows the helix number and their relative order (2-3-1-4) similar to what is …
Figure 4—figure supplement 3
Establishment of LLC-sh-TLR4 cells and analysis of SCGB3A2/LPS binding/incorporation.
(A) Assessment of shRNA knockdown efficiency in LLC Cells. LLC cells were transfected with sh-TLR4 plasmid using retrovirus vector and stable cell clones were prepared. Each clone was grown in 2% …
Figure 4—figure supplement 4
Effect of SCGB3A2-LPS on RAW264.7 cells.
(A) Flow cytometric analysis for SDC1 expression on RAW264.7 cells. PE conjugated mouse SDC1 ectodomain specific antibody was used for detection of cell surface SDC1 expressions on RAW264.7 cells. (B…
Figure 4—figure supplement 5
Confirmation of caspase-11 knockdown in LLC cells in the presence of Wedelolactone or LLC-sh-casp11 cells.
(A) Immunoblots of LLC cells for caspase-11 and GAPDH after treatment with or without LPS (L, 10 ng/ml), SCGB3A2 (S, 200 ng/ml), Wedelolactone (W, 50 µM) in 2%FBS-RPMI for 3 hr. C: control. (B) Immun…
Figure 5
SCGB3A2-LPS promotion of pyroptotic cell death.
(A) Representative phase contrast image of LLC cells incubated with LPS (O111:B4, 1 µg/ml) and SCGB3A2 (1 µg/ml) in 1% FBS-RPMI 1640 for 72 hr. Arrows indicate cells undergoing pyroptosis (swollen …
Figure 6
Evaluation for the requirement of SDC1 and other genes for SCGB3A2-LPS effect.
(A) CCK8 assay using LLC-sh-Control and LLC-sh-SDC1 for 72 hr in 1% FBS-RPMI 1640 medium. C; control, S; human SCGB3A2 (200 ng/ml), L; LPS (O111:B4, 1 pg/ml). Averages ± SD from more than three …
Tables
Key resources table
| Reagent type (species) Or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | Scgb3a2-/- (C57BL/6N background) | In house | Kido et al., 2014 | |
| Strain, strain background (M. musculus) | Scgb3a2fl/fl (C57BL/6N background) | In house | Kido et al., 2014 | |
| Strain, strain background (M. musculus) | Ccsp-Cre;LSL-K-rasG12D (mixed background) | Francesco DeMayo (Baylor College of Medicine, now NIEHS) | Moghaddam et al., 2009 | |
| Cell line (M. musculus) | LLC-Mhi | Glenn Merlino (NCI) | Highly metastatic, MAP test negative | |
| Cell line (M. musculus) | B16F10 | ATCC | CRL-6475 | |
| Cell line (M. musculus) | RAW264.7 | Raymond Donnelly (FDA) | MAP test negative, authenticated by STR | |
| Cell line (human) | ARH-77 ARH-77-mSDC1 | Ralph Sanderson (The University of Alabama at Birmingham) | MAP test negative, authenticated by STR | |
| Recombinant protein | mouse SCGB3A2 | NCI Protein Expression Core | Kurotani et al., 2008 | |
| Recombinant protein | mouse SCGB3A2 | Hölzel Diagnostics | CSB-BP846028MO | |
| Recombinant protein | mouse SCGB3A2 | CosmoBio USA | CSB-EP846028MO | |
| Recombinant protein | human SCGB3A2 | In house (APCBio Innovations) | Cai et al., 2014 | |
| Antibody | anti-mouse SCGB3A2 (rabbit polyclonal) | E. Coli expressed mature SCGB3A2 in vector pET-32a(+) used as an antigen to produce a polyclonal antibody (produced by Macromolecular Resources, Fort Collins, CO) | Niimi et al., 2001 | 1:5000 for IHC |
| Antibody | anti-human SCGB3A2 (polyclonal) | In house (APCBIo Innovations) | Cai et al., 2014 | 1:1000 for WB |
| Antibody | anti-mouse/human SDC1 | Pynog W Park (Harvard Med Sch) | 1:200 for IHC/IF 1:1000 for WB | |
| Antibody | PE-rat anti-mouse SDC1 | BD Pharmingnen | clone 281.2 | 1:200 for FACS |
| Antibody | anti-clathrin heavy chain | Cell Signaling Technology | P1663 | 1:200 for IF |
| Antibody | anti-EEA1 (monoclonal) | Cell Signaling Technology | C45B10 | 1:200 for IF |
| Antibody | anti-IL-1β (monoclonal) | Cell Signaling Technology | Clone 3A6 | 1:1000 for WB |
| Antibody | anti-caspace-11 (monoclonal) | Thermo Fisher Scientific | Clone 17D9 | 1:1000 for WB 1:100 for IHC/IF |
| Antibody | anti-ICAM1 (monoclonal) | Thermo Fisher Scientific | MA5407 | 1:200 for IF |
| Antibody | anti-caspace-1 (monoclonal) | Thermo Fisher Scientific | Clone 5B10 | 1:1000 for WB 1:100 for IF |
| Antibody | anti-NLRP3 (monoclonal) | Adipogen | AG-20B-0014 | 1:1000 for WB 1:200 for IHC/IF |
| Antibody | anti-LAMP1 (monoclonal) | Santa Cruz | sc-17768 | 1:200 for IF |
| Antibody | anti-HaloTag (polyclonal) | Promega | G9281 | 1:100 for IF 1:1000 for WB |
| Antibody | anti-GAPDH (monoclonal) | Proteintech | 60004–1-Ig | 1:5000 for WB |
| Antibody | anti-LPS (monoclonal) | Abcam | ab35654 | 1:1000 for WB |
| Chemical compound, drug | LPS from E. coli O111:B4 | Sigma-Aldrich | L4391 | |
| Chemical compound, drug | Ra mutant LPS from E.coli EH-100 | Sigma-Aldrich | L9641 | |
| Chemical compound, drug | LPS from E.coli K-235 | Sigma-Aldrich | L2018 | |
| Chemical compound, drug | LPS from Salmonella typhimurium | Sigma-Aldrich | L2262 | |
| Chemical compound, drug | heparin sodium salt from porcine intestinal mucosa | Sigma-Aldrich | H3393 | |
| Chemical compound, drug | imidazole | Sigma-Aldrich | I5513 | |
| Chemical compound, drug | zinc sulfate solution | Sigma-Aldrich | Z2876 | |
| Chemical compound, drug | Dynasore | AdooQ Bioscien | A12726 | |
| Chemical compound, drug | Wedelolactone | AdooQ Bioscien | A14804 | |
| Chemical compound, drug | LPS-EB Biotin | InvivoGen | tlrl-3blps |
Additional files
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Supplementary file 1
LPS contents in various preparations/batches of recombinant SCGB3A2
- https://doi.org/10.7554/eLife.37854.017
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Supplementary file 2
116 proteins identified as those significantly bound to human SCGB3A2 in protein array.
Proteins with Z-Factor >0.9, Z-Score >4.0 are listed. High score indicates a strong protein-protein interaction.
- https://doi.org/10.7554/eLife.37854.018
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Supplementary file 3
SCGB3A2 candidate receptors identified by human protein interaction array
- https://doi.org/10.7554/eLife.37854.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.37854.020
