Journal: Cancer Management and Research

Article Title: Distribution of CD8 T Cells and NK Cells in the Stroma in Relation to Recurrence or Metastasis of Nasopharyngeal Carcinoma

doi: 10.2147/CMAR.S365230

Figure Lengend Snippet: CD8 T cells, NK cell, macrophages, and plasma cell were stained in tumor core and stromal in NPC by immunohistochemistry. ( A ). CD8 200× ( B ). CD8 400× ( C ). CD56 200× ( D ). CD56 400× ( E ). CD68 200× ( F ). CD68 400× ( G ). CD138 200× ( H ). CD138 400×.

Article Snippet: These TIICs were stained with different antibodies as follows: an anti-human CD8 mouse monoclonal antibody (TA802079S, dilution 1:150, Origene), an anti-human CD56 mouse monoclonal antibody (TA805376S, dilution 1:150, Origene), an anti-human CD68 mouse monoclonal antibody (TA802949S, dilution 1:500, Origene), and an anti-human CD138 mouse monoclonal antibody (TA813799S, dilution 1:1000, Origene).

Techniques: Clinical Proteomics, Staining, Immunohistochemistry

Journal: eLife

Article Title: A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death

doi: 10.7554/elife.37854

Figure Lengend Snippet: Figure 3. 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. IP and western blotting were sequentially carried out using anti-FLAG and anti-Myc antibody, respectively. (C) SCGB3A2 and SDC1 immunostaining in the airway epithelial cells of adult wild-type mouse lungs. Counterstained with Hematoxylin. Bar = 10 mm. (D) Immunofluorescent staining of SDC1 in LLC and B16F10 cells grown in 10%FBS-RPMI 1640 medium for 24 hr. DAPI was used for nuclear staining. White arrow indicates SDC1 cell surface expression in LLC. Red arrowhead points to the staining at cell-cell junction and white arrowheads point to focal SDC1 staining near the nucleus in B16F10 cells. Bar = 20 mm. (E) Flow cytometric analysis for SDC1 expression on cell surfaces of LLC and B16F10 cells using PE conjugated anti-SDC1 ectodomain specific antibody. (F) Flow cytometric analysis for SCGB3A2 binding to LLC cells using anti-SCGB3A2 antibody. GST tagged mouse SCGB3A2 (3 mg) was incubated with LLC cells at 4 ˚C for 30 min. Cells were stained with rabbit anti-mouse SCGB3A2 antibody, followed by staining with PE-anti-rabbit IgG antibody at 4 ˚C for 30 min. (G) SCGB3A2 binding assay on LLC-sh-Control or sh-SDC1 cells. GST tagged mouse SCGB3A2 (1 mg) was incubated with each cell type at 4 ˚C for 30 min, followed by staining with Alexa 488 anti-rabbit IgG antibody at 4 ˚C for 30 min. (H) SCGB3A2 binding assay on LLC cells. Cells were co- incubated with or without GST tagged mouse SCGB3A2 (1 mg) or SCGB3A2 + heparin. Cells were stained with PE-anti-rabbit IgG antibody at 4 ˚C for 30 min. Data except A are the representative from more than three independent experiments. DOI: https://doi.org/10.7554/eLife.37854.006 The following source data and figure supplement are available for figure 3:

Article Snippet: The shRNA constructs were purchased from transOMIC for mouse SDC1, from ORIGENE for mouse TLR4 and mouse caspase-11.

Techniques: Binding Assay, Protein Array, Isolation, Co-Immunoprecipitation Assay, Western Blot, Immunostaining, Staining, Expressing, Incubation, Control

Journal: eLife

Article Title: A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death

doi: 10.7554/elife.37854

Figure Lengend Snippet: Figure 4. 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 without GST- mSCGB3A2 (1 mg). Scale bar = 10 mm. (B) Immunofluorescence analysis of LLC cells for clathrin and HT (HaloTag). Cells were incubated with LPSA488 (1 mg/ml) and SCGB3A2-HT supernatant (SN) in 0%1 hr. Arrowheads: uropod structure. Bar = 10 mm. (C) SCGB3A2 tetramer model (see Figure 4—figure supplement 2A–2F). Left: Exploded view of the tetramer model showing the two dimers, which are colored to identify the two monomers. The dimer structure reveals a pocket accessible from Face A and flanked by positively charged residues (Gly 1 N termini-; Arg 6 and Lys 61) forming a pattern Figure 4 continued on next page

Article Snippet: The shRNA constructs were purchased from transOMIC for mouse SDC1, from ORIGENE for mouse TLR4 and mouse caspase-11.

Techniques: Immunofluorescence, Incubation

Journal: eLife

Article Title: A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death

doi: 10.7554/elife.37854

Figure Lengend Snippet: 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 independent experiments, each in triplicate are shown. *: p<0.05 by one-way ANOVA. (B) Immunofluorescent staining of caspase-11 and LPSA594 using LLC-sh-Control and LLC-sh-SDC1 cells. Cells were maintained in 1% FBS-RPMI 1640 medium for 16 hr with or without human SCGB3A2 (1 mg/ml), LPS Figure 6 continued on next page

Article Snippet: The shRNA constructs were purchased from transOMIC for mouse SDC1, from ORIGENE for mouse TLR4 and mouse caspase-11.

Techniques: CCK-8 Assay, Control, Staining

Journal: Frontiers in Oncology

Article Title: Differential Impact of Membrane-Bound and Soluble Forms of the Prognostic Marker Syndecan-1 on the Invasiveness, Migration, Apoptosis, and Proliferation of Cervical Cancer Cells

doi: 10.3389/fonc.2022.803899

Figure Lengend Snippet: Sdc-1 is dysregulated in cervical carcinoma, and its high expression correlates with poor overall survival. Boxplots of SDC-1 gene expression in cervical cancer tissue when comparing paired normal and tumor RNA Seq data of three patients. (A) and when comparing normal, tumor and metastasis RNA Seq data (B) . The quantile cutoff values (minimum, 1st quartile, median, 3rd quartile, maximum) and the number of analyzed samples are presented. (C) Kaplan-Meier Survival analysis of 304 cervical carcinoma patients stratified by high or low expression of Sdc-1. (A) p=0.181 (not significant), Mann-Whitney-Test. (B) p=0.069 (Kruskal-Wallis test, trend for significance), post hoc -analysis p-values (Dunn’s test): p=1.65x10 -03 normal tissue compared to tumors (significant), p=0.175 tumor tissue compared to metastases (not significant), p=0.115 normal tissue compared to metastatic tissue. (C) p=0.024 (significant), Cox regression analysis log rank p-value.

Article Snippet: Cells were then stained with rat-anti-mouse Sdc-1 mAb 281-2 (BD Pharmingen, San Jose, CA, 1:1000 in PBS/1% bovine serum albumin (BSA)), Endogenous peroxidase was quenched with methanol/0.6% H 2 O 2 , followed by three washes with PBS.

Techniques: Expressing, RNA Sequencing Assay, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Differential Impact of Membrane-Bound and Soluble Forms of the Prognostic Marker Syndecan-1 on the Invasiveness, Migration, Apoptosis, and Proliferation of Cervical Cancer Cells

doi: 10.3389/fonc.2022.803899

Figure Lengend Snippet: Characterisation of HeLa cells overexpressing wild-type, constitutively membrane bound (Sdc-388) and constitutively shed (Sdc1-392) Sdc-1. (A) Schematic representation of the plasmid Sdc-1 cDNA inserts, CMV, cytomegalovirus promoter; yellow box, juxta-membrane domain; Black box, non-cleavable CD4 sequence; blue box, transmembrane and cytoplasmic domain, orange box, poly-A-tail; Sdc1-WT murine wild-type form; Sdc1-388, uncleavable construct 388;Sdc1-392, constitutively shed construct 392. (B) Quantitative PCR analysis of murine (mu Sdc-1 and human (Hu Sdc-1) Sdc1 expression. Sdc-1 expression was related to the housekeeping gene 18SrRNA. n ≥ 3, error bars = SEM. (C) Detection of human Sdc-1 protein expression at the surface of the transfected HeLa cell lines using flow cytometry. Cells were stained for isotype control mouse IgG1-PE and mouse anti-human Sdc-1 (CD138)-PE and the cells were subjected to flow cytometry. Human Sdc-1 is expressed at the cell surface of all cell types. (D) Immunocytochemistry for murine Sdc-1, demonstrating expression of murine Sdc-1 in Sdc1-WT, Sdc-1-388 and Sdc-1 392 transfected cells (brown-red staining). Original magnification 10x. (E, F) Detection of shed human (E) and murine (F) Sdc-1 in cell culture supernatants of the transfected cell lines. Conditioned media were collected from the cell lines indicated and 600 µl were subjected to a dotblot assay and quantified by Image J densitometric analysis. Left panels = quantification, right panels= representative dot-blots, n>3, *= p<0.05 Sdc1-392 compared to vector control (t-test). The cell lines shed comparable amounts of human Sdc-1 into the culture media, with a moderately, yet significantly enhanced amount in Sdc1-392 cells. Shed amounts of murine Sdc-1 were variable, with Sdc1-WT cells showing significantly increased levels of shed murine Sdc-1 compared to vector control (n>3, *p<0.05, t-test).

Article Snippet: Cells were then stained with rat-anti-mouse Sdc-1 mAb 281-2 (BD Pharmingen, San Jose, CA, 1:1000 in PBS/1% bovine serum albumin (BSA)), Endogenous peroxidase was quenched with methanol/0.6% H 2 O 2 , followed by three washes with PBS.

Techniques: Plasmid Preparation, Sequencing, Construct, Real-time Polymerase Chain Reaction, Expressing, Transfection, Flow Cytometry, Staining, Immunocytochemistry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Differential Impact of Membrane-Bound and Soluble Forms of the Prognostic Marker Syndecan-1 on the Invasiveness, Migration, Apoptosis, and Proliferation of Cervical Cancer Cells

doi: 10.3389/fonc.2022.803899

Figure Lengend Snippet: Role for soluble and membrane bound forms of Sdc-1 in HeLa cell proliferation, cell cycle progression, apoptosis and invasion. (A) Differential effect of membrane-bound and soluble Sdc-1 on breast cancer cell proliferation. Control vector-transfected HeLa and HeLa cells stably overexpressing WT (Sdc1-WT), constitutively membrane-bound (Sdc1-388) or the soluble ectodomain (Sdc1-392) of Sdc1 were subjected to an Alamar Blue cell proliferation assay * P < 0.05 (One-way ANOVA with Dunn’s post hoc test) for Sdc1-388 compared to vector control and Sdc1-392 compared to vector control n ≥ 3, error bars = SEM. Changes in apoptosis (B) and cell cycle progression (C) after the stable transfection of HeLa cells as quantified by using Annexin V/propidium iodide (B) and by DNA staining (C) and followed by flow cytometry. * P < 0.05 (One-way ANOVA with Dunn’s post hoc test) for Sdc1-388 compared to vector control, and Sdc1-392 compared to vector control, n ≥ 3, error bars = SEM. (D) quantitative Real-Time PCR of the expression of the apoptosis markers Bad, Bak and Bcl-2 in the HeLa Sdc1-388 transfected cells. Data are expressed as fold change versus control vector-transfected cells. ns, no significant p value for all group comparisons (one-way ANOVA with Dunn’s post hoc test). n≥3, error bars = SEM. (E) Stably transfected HeLa cells were subjected to a matrigel invasion assay. Quantification of invasive cells relative to control vector-transfected cells. *p <0.05 Sdc1-WT compared to vectror controls and SDc1-392 compared to vector controls, (one-way ANOVA with Dunn’s post hoc test), n≥4, error bars = SEM. (F) quantitative RT-PCR analysis of E-cadherin, MMP2 and TIMP1 compared with vector controls. *p <0.05, MMP2 Sdc1-388 compared to vector controls, E-cadherin Sdc1-388 compared to vector controls, ***p <0.001, E-cadherin Sdc1-392 compared to vector controls (one-way ANOVA with Dunn’s post hoc test), n≥3, error bars = SEM.

Article Snippet: Cells were then stained with rat-anti-mouse Sdc-1 mAb 281-2 (BD Pharmingen, San Jose, CA, 1:1000 in PBS/1% bovine serum albumin (BSA)), Endogenous peroxidase was quenched with methanol/0.6% H 2 O 2 , followed by three washes with PBS.

Techniques: Plasmid Preparation, Transfection, Stable Transfection, Proliferation Assay, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Invasion Assay, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Differential Impact of Membrane-Bound and Soluble Forms of the Prognostic Marker Syndecan-1 on the Invasiveness, Migration, Apoptosis, and Proliferation of Cervical Cancer Cells

doi: 10.3389/fonc.2022.803899

Figure Lengend Snippet: Migration of HeLa cells is decreased by all forms of Sdc-1 in a Rho-GTPase dependent manner. (A) The Scratch/wound area of the four cell lines is shown as a percentage of the wound area at time 0h, 6h, 8.5h and 23h. The ability of all three cell constructs to migrate is reduced compared to the control cell line. * = p <0.05 (nonparametric Friedman’s test with Dunn’s posttest) for Sdc1-388 compared to vector control (t=6h, 8.5h, 23h), for Sdc1-WT vs vector control (t=23h) and for Sdc1-392 vs vector control (t=6h, 23h), n≥3, error bars = SEM. (B) Stably transfected HeLa cells were treated with the Rho kinase (ROCK) inhibitor Y-27632 and then a Scratch/wound area was created. The area of the three cell constructs and vector was quantified at 0h, 6h, 8.5h and 23h. ns, no significant difference compared to vector control (nonparametric Friedman’s test with Dunn’s posttest). n≥3, error bars = SEM. (C) Confocal immunofluorescence microscopic analysis of RhoB protein in HeLa transfected cells. green, RhoB; red, actin-binding protein phalloidin for cytoskeletal staining; blue, DAPI staining for nucleus. Representative images are presented. 40x magnification.

Article Snippet: Cells were then stained with rat-anti-mouse Sdc-1 mAb 281-2 (BD Pharmingen, San Jose, CA, 1:1000 in PBS/1% bovine serum albumin (BSA)), Endogenous peroxidase was quenched with methanol/0.6% H 2 O 2 , followed by three washes with PBS.

Techniques: Migration, Construct, Plasmid Preparation, Stable Transfection, Transfection, Immunofluorescence, Binding Assay, Staining