Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: CST6 suppresses osteoclastogenesis and bone metastasis. ( A ) Osteoclastogenesis of murine primary bone marrow cells with conditioned medium (CM) from CST6 -overexpressing or control SCP2 (n = 3 independent experiments). Ctrl, control. ( B ) Osteoclastogenesis of primary bone marrow culture with CM from CST6 knockdown or control SCP4 cells (n = 3 independent experiments). ( C-F ) Intracardiac injection of SCP2 cells with CST6 overexpression for bone metastasis analysis (n = 10 mice per group). Shown are representative bioluminescence imaging (BLI), micro-CT, H&E and TRAP staining of the hind legs ( C ), ex vivo BLI analysis of hind legs of the mice ( D ), BLI quantitation of limb metastasis ( E ) and quantitation of TRAP + cells along the tumor-bone interface ( F ). White and black arrows ( C ) point to the area of bone damage and osteoclasts, respectively. Scale bar, 200 μm. ( G ) Intracardiac injection of SCP4 cells with CST6 knockdown for bone metastasis analysis. ( H ) Osteoclastogenesis of primary bone marrow cells treated with 32 nM human or mouse (mCST6) recombinant CST6 proteins. ( I ) Osteoclastogenesis of RAW264.7 cells cultured with RANKL and treated with 32 nM human CST6 protein or 32 nM human CST6 and 7 µg/ml CST6 neutralizing antibody. ( J ) Expression of various gene markers of osteoclast differentiation in RAW264.7 cells treated with CST6 recombinant protein or CST6 recombinant protein and CST6 neutralizing antibody (n = 3 biological repeats). Scale bar, 150 μm. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Control, Knockdown, Injection, Over Expression, Imaging, Micro-CT, Staining, Ex Vivo, Quantitation Assay, Recombinant, Cell Culture, Expressing

Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: CST6 regulates osteoclastogenesis by inhibiting CTSB. ( A ) CTSB enzymatic activity in lysates of SCP2 cells expressing wild type or mutant CST6 (n = 3 independent experiments). ∆N, N64A; ∆W, W135A; ∆NW, N64A and W135A double mutant. ( B, C ) Osteoclastogenesis of primary bone marrow treated with CM from SCP2 overexpressing CST6 mutants (n = 3 independent experiments). Representative images of TRAP staining were shown in C . Scale bar, 150 μm. ( D, E ) In vivo bone metastasis analysis of SCP2 cells expressing CST6 mutants (n = 10 mice per group). Shown are representative images of BLI, H&E and TRAP staining of bone metastases in hind legs at week 6 after SCP2 injection ( D ), and quantitation of BLI signal and TRAP + cells ( E ). Scale bar, 200 μm. ( F ) Osteoclastogenesis of primary bone marrow treated with 10 μM Z-FY(t-Bu)-DMK (Z-FY) or CA-074Me (n = 3 independent experiments). ( G ) RAW264.7 osteoclastogenesis after treatment with Z-FY or CA074Me (n = 3 independent experiments). ( H ) Osteoclastogenesis of murine primary bone marrow cells transfected with Ctsb siRNA. ( I ) Intracellular CTSB activity of RAW264.7 after treatment with 32 nM CST6 protein. ( J ) RAW264.7 was cultured with recombinant His-tagged CST6 protein for the indicated time, and intracellular CST6-His level was analyzed by Western blots after PBS washing of the cells. α-Tub, α-Tubulin. ( K ) Western blot analysis of intracellular CST6-His level of RAW264.7 after culturing the cells with CST6-His protein and various concentrations of Dynasore. ( L ) RAW264.7 osteoclastogenesis after CST6 protein and Dynasore treatment. Scale bar, 150 μm. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Activity Assay, Expressing, Mutagenesis, Staining, In Vivo, Injection, Quantitation Assay, Transfection, Cell Culture, Recombinant, Western Blot

Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: CST6 regulates osteoclastogenesis by stabilizing SPHK1 and inhibiting p38. ( A ) SPHK1 and phosphorylation of p38 in murine primary bone marrow cells transfected with Ctsb siRNA. ( B ) SPHK1 and phosphorylation of p38 in RAW264.7 after treatment with CA074Me or CST6 protein. ( C ) RAW264.7 osteoclastogenesis after Sphk1 overexpression and Sphk1 knockdown. ( d-f ) SPHK1 expression ( D ) and osteoclastogenesis ( E, F ) of murine primary bone marrow cells with Ctsb and Sphk1 knockdown. ( G, H ) SPHK1 expression ( G ) and osteoclastogenesis ( H ) of RAW264.7 after Sphk1 knockdown and recombinant CST6 treatment. ( I ) Phosphorylation of p38 in primary bone marrow cells with Ctsb knockdown (left) and in RAW264.7 cells after Sphk1 knockdown (right), together with RANKL treatment of various time. ( J ) Phosphorylation of p38 in RAW264.7 cells treated with CM from CST6 -ovexpressing or control SCP2 cells, together with RANKL treatment of various time. ( K, L ) Phosphorylation of p38 ( K ) and osteoclastogenesis ( L ) of RAW264.7 treated with RANKL, SB203580 and CM from CST6 -knockdown or control SCP4 cells. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Phospho-proteomics, Transfection, Over Expression, Knockdown, Expressing, Recombinant, Control

Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: CST6 recombinant protein effectively inhibits bone metastasis of breast cancer in mice. ( A ) Representative BLI, micro-CT, H&E and TRAP staining images of SCP2 bone metastases at week 5 after treatment of 1 mg/kg/day wild type or mutant CST6 proteins (n = 10 mice per group). Scale bar, 200 μm. ( B, C ) BLI ( B ) and osteoclast ( C ) quantitation of the metastasis. ( D ) Survival analysis of the mice. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Recombinant, Micro-CT, Staining, Mutagenesis, Quantitation Assay

Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: CST6 peptides inhibit osteoclastogenesis and bone metastasis of breast cancer. ( A ) The effects of CST6 peptides to inhibit CTSB activity of RAW264.7 lysate (n = 3 independent experiments). ( B ) Osteoclastogenesis of primary bone marrow (n = 3 independent experiments) treated CST6 peptides or Zoledronic Acid (ZA). Scale bar, 150 μm. Asterisks indicate significance compared to the control. ( C-G ) The effects of CST6 peptides to inhibit SCP2 bone metastasis in mice after treatment of 1 mg/kg/day CST6 peptides (n = 10 mice per group). Shown are representative BLI, micro-CT, H&E and TRAP staining images of bone metastases in hind legs at week 5 ( C ), BLI quantitation of metastasis ( D ), TRAP + cell quantitation in metastases ( E ), body weights ( F ) and survival analysis ( G ) of the mice. Scale bar, 200 μm. ( H, I ) SCP2 bone metastasis in nude mice ( H ) and animal survival ( I ) with treatment of 1 mg/kg/day CST6 peptides, 1 mg/kg/day Zoledronic Acid (ZA) or 1 mg/kg/day Bortezomib (BO). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Activity Assay, Control, Micro-CT, Staining, Quantitation Assay

Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: Half-life and toxicity analysis of CST6 protein and peptides in mice. ( A ) Half-life analysis of CST6 protein and peptides in the blood of mice. ( B ) Body weights of mice 4 weeks after daily treatment of 1 mg/kg/day CST6 protein and peptides (n = 4 mice per group). ( C ) H&E staining of various organs in mice 4 weeks after daily treatment of 1 mg/kg/day CST6 protein and peptides. Scale bar, 200 μm.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Staining

Journal: Theranostics

Article Title: CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis

doi: 10.7150/thno.62187

Figure Lengend Snippet: Hematological analysis of mice after treatment of CST6 protein or peptides. Shown are the hematological results after intravenous treatment of 1 mg/kg/day CST6 protein and peptides for 4 weeks. WBC, white blood cell; RBC, red blood cell; MCV, erythrocyte mean corpuscular volume; HGB, hemoglobin; MCH, mean corpuscular hemoglobin; HCT, red blood cell specific volume; MCHC, mean corpuscular hemoglobin concentration.

Article Snippet: The rabbit anti-CST6 polyclonal antibody (17076-1-AP, Proteintech), mouse anti-SPHK1 monoclonal antibody (365401, Santa Cruz), rabbit anti-phos-p38 (Thr180/Tyr182) polyclonal antibody (9211, Cell Signaling Technology) and rabbit anti-p38 polyclonal antibody (9212, Cell Signaling Technology) were used for Western blotting.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Expression and Regulation of a Novel Decidual Cells-Derived Estrogen Target during Decidualization

doi: 10.3390/ijms24010302

Figure Lengend Snippet: Cstb expression is associated with angiogenesis in mouse uterine stromal cells Angptl7 . ( A ) Gene ontology (GO) functional classification of the DEGs. ( B ) The expressions of Cstb and ZO1 were detected by immunofluorescence in the uterus on day 6. Bar = 300 μm. ( C ) The expression of Angptl7 in stromal cells of the con NC group, dc NC group, and dc si Cstb group was detected by RT-qPCR. ( D ) The expression of Angptl7 in mouse uteri from days 5 to 8 of pregnancy was detected by in situ hybridization. Bar = 300 μm. * p < 0.05. NC, negative control; si Cstb , siRNA of Cstb ; dc, in vitro decidualization.

Article Snippet: Briefly, frozen sections were fixed in 4% paraformaldehyde solution for 10 min and then soaked in 0.1% Triton X-100 in PBS for 15 min. After blocking with 5% donkey serum (Zhongshan Jinqiao, Beijing, China) in a 37 °C oven for 60 min, the sections were incubated with mouse anti- Cstb (1:500, Santa Cruz, sc-166561), ZO1 (1:200, Cell Signaling Technology, Boston, MA, USA, #13663) diluted in PBS overnight in a 4 °C refrigerator.

Techniques: Expressing, Functional Assay, Immunofluorescence, Quantitative RT-PCR, In Situ Hybridization, Negative Control, In Vitro

Journal: Cells

Article Title: Stefin B and Cystatin C Deficiency Suppresses Tumor Growth and Alters Tumor Microenvironment in a Breast Cancer Model

doi: 10.3390/cells15040360

Figure Lengend Snippet: Expression of stefin B and cystatin C, and cysteine cathepsin activity, in PyMT mammary tumors of different genotypes. ( A ) Western blot for stefin B (StfB) in mammary tumors from wild-type (PyMT;WT; n = 3), stefin B knockout (PyMT; StfB −/− ; n = 3), cystatin C knockout (PyMT; CstC −/− ; n = 3), and double-knockout (PyMT;DKO; n = 3) mice at 14 weeks of age. StfB is absent in PyMT; StfB −/− and PyMT;DKO tumors, confirming successful knockout. β-actin was used as a loading control. ( B ) Western blot for cystatin C (CstC) in the same tumor samples. CstC is absent in PyMT; CstC −/− and PyMT;DKO tumors, verifying successful knockout. β-actin was used as a loading control. ( C ) Relative cathepsin activity determined in homogenates of PyMT tumors (PyMT;WT, n = 4; PyMT;DKO, n = 3). Data represent biological replicates from independent tumors derived from different mice. Activity was measured as Z-Phe-Arg-AMC hydrolysis in the presence or absence of E64. Differences were analyzed using Student’s t -test. ( D ) Relative cathepsin activity determined in cell culture medium of primary tumor cells (PyMT;WT, n = 3; PyMT;DKO, n = 3). Data represent biological replicates from independent primary tumor cell isolates derived from different mice. Activity was measured as Z-Phe-Arg-AMC hydrolysis in the presence or absence of E64. Differences between groups were analyzed using Student’s t -test.

Article Snippet: Membranes were probed with anti-CstC antibody (Abcam, Cambridge, UK; ab109508, 1:100,000) and anti-StfB antibody (R&D Systems, Minneapolis, MN, USA; MAB1409, 1:5000).

Techniques: Expressing, Activity Assay, Western Blot, Knock-Out, Double Knockout, Control, Derivative Assay, Cell Culture

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 1. Phenotype of rescued Tg(INV-Cst6)Cst6ichq/ichq mice. A) Tg(INV-Cst6)Cst6ichq/ichq mice survived and showed periodic hair loss. After 4 mo the progenies became completely bald. Keratitis and thickening of the cornea were observed in Tg(INV-Cst6)Cst6ichq/ichq mice from 4 to 5 mo. The mice shown are 9 and 32 wk old. Inset: magnified view of an affected eye. B) Keratitis and metaplasia of the corneal epithelium in Tg(INV- Cst6)Cst6ichq/ichq mice. H&E staining of the eye and the cornea in WT and Tg(INV-Cst6)Cst6ichq/ichq mice. C) Immunofluores- cence staining for the expression of loricrin (LOR) and filaggrin (FLG) in the cornea. Scale bars, 100 mm.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Staining, Expressing

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 2. Destruction of the HFs in Tg(INV-Cst6)Cst6ichq/ichq mice. A) Immunofluorescence double labeling of INV and Cst6 in the epidermis and the HF of WT mice. Cst6 was expressed in the stratum granulosum and halfway up the HF, including the area around the bulge, whereas INV expression was also seen in the stratum granulosum, but remained only in the proximal part of the HF. B) Immunohistological labeling in WT mice to detect the location of the bulge area (arrows) using the stem cell markers CD34 and keratin 15 and the proliferation marker Ki67. C) H&E staining of Tg(INV-Cst6)Cst6ichq/ichq mice from 11, 13, and 16 wk showed disappearance of the HFs. Scale bar, 100 mm.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Labeling, Expressing, Marker, Staining

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 3. Inhibition of mouse CtsB by mouse Cst6. A) BMV109 labels active cysteine cathepsins, such as CTSB and CTSX (14) in RAW cell lysates (murine Mf cell line). Lane 1, no inhibitor; lane 2, JPM-OEt (pan-cathepsin inhibitor, 50 mM); lane 3, CA-074 (CtsB inhibitor, 10 mM); lane 4, Z-FY (t-BU)DMK (CtsL inhibitor, 10 mM); lane 5, mouse Cst6 (18 mM); and lane 6, heat inactivation. Note that CA-074 and Cst6 inhibited CtsB (lane 3 and 5). M = precision plus protein dual-color marker. B) The Ki for the inhibition of CtsB by Cst6 was determined by measuring the residual enzymatic activity of a fixed concentration of enzyme, incubated with increasing concentrations of the inhibitor. An Easson-Stedman plot (inset) was used to calculate the Ki, according to the following equation: [I]/1 2 a = (Ki/a) + E0, were I is the inhibitor concentration, E0 is the enzyme concentration at time 0, and a is the fractional activity. The plot yielded a straight line with a Ki slope of 0.98 nM.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Inhibition, Chromosome Transmission Fidelity Colony Color Assay, Activity Assay, Concentration Assay, Incubation

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 4. CtsB and Cst6 colocalize in the mouse HF. A) Immunofluorescence double staining for Cst6 (green) and CtsB (red) in WT mice revealed that colocalization of both proteins was found in the proximal part of the HF as well as the lower region where the bulge area resides. Cst6 is also expressed in the epidermis, whereas no CtsB is observed. Scale bar, 100 mm. B) Cst6 and CtsB expression in mouse epidermis and HFs of Tg(INV-Cst6)Cst6ichq/ichq mice. No immunofluorescence staining for Cst6 was observed in the lower region of the HF (close to the bulge, arrowheads). Scale bar, 100 mm. C) Schematic presentation of INV, Cst6, and CtsB localization in the HF and epidermis of WT mice and the situation in rescued transgenic Tg(INV-Cst6)Cst6ichq/ichq mice.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Double Staining, Expressing, Staining, Transgenic Assay

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 5. Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade (corneo)-desmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3) The findings in the present study suggest that inhibition of CtsB by CST6 protects HF maintenance in mice. 4) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse CST6.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Activity Assay, Control, Inhibition, Functional Assay

Journal: Journal of Extracellular Vesicles

Article Title: Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses

doi: 10.1002/jev2.12071

Figure Lengend Snippet: Human milk EVs inhibit agonist‐induced activation of endosomal TLR3 and TLR9, but not of cell surface TLR2 and TLR4. [a‐d) Secretion of SEAP reporter protein was determined for TLR reporter cell lines cultured in indicated conditions either or not in response to agonist (Pam3CSK for TLR2, LPS for TLR4, Poly I:C for TLR3, and CpG ODN2006 for TRL9), with the TLR‐specific agonist set to 100%. Box and whisker plots contain data from a single technical replicate of three independent experiments with a total of four different milk donors. e) Relative gene expression of IL6 and CXCL8 in Ca9‐22 cells cultured for 5 h with medium, EV, Poly I:C or Poly I:C + EV. Delta Ct‐values to ACTB and GAPDH were calculated and expressed relative to medium controls. EVs from two different milk donors were used and PCR reaction was performed twice on a single technical replicate. Results are summarized in bar graphs as mean ± SD. f) Heatmap representing the cellular gene expression profile of Ca9‐22 cells cultured for 4 h in the four different conditions (medium, agonist, EV + agonist, EV‐depleted + agonist) in a gradient running from minimal gene expression (green) to maximal expression (red) for each gene analyzed (see Supplementary for complete dataset). Data is from 1 technical replicate derived from 1 experiment, with 1 milk donor. g) Schematic representation of key signaling pathways involved in TLR signaling of the individual TLRs tested which are shown in one Figure. TLR2, TLR4 and TLR9 signal via MyD88, while TLR3 signals via TRIF (not shown) to NF‐kB (via IKK) and/or AP‐1 leading to cytokine production. TLR2 and TLR4 are surface receptors, while TLR3 and TLR9 are sorted into the endosome where they are cleaved which enhances their signaling. Proteins from milk EVs are depicted in blue, while interacting cellular proteins are shown in white. Type of interactions between proteins is either shown as activating or inhibiting. In every assay, the individual TLR was activated via its specific ligand, which is shown in red. Note that TLR2 is present in milk EVs as well as in the TLR2 reporter cells. h) Western blot analysis for the presence of Cystatin‐B (CSTB; expected size 11 kDa; exposure time 19 s) in purified milk EVs or in EV‐depleted control. Data is from one experiment with a single sample from nine different milk donors. i) Western blot analysis for the presence of TLR3 on whole cell lysate of Ca9‐22 cells cultured in medium alone or stimulated with poly I:C with or without milk EVs or EV‐depleted control. Full length (FL) TLR3 (expected size 130 kDa) and cleaved TLR3 (expected size 70 kDa) are visible. GAPDH (expected size 36 kDa) was used as a loading control and applied to normalize the intensity of the FL band and cleaved band (shown as volume intensity below the blot) in order to quantitatively compare signals. A total of three different milk donors were tested in a single experiment. Significance was calculated with 2‐way mixed model analysis and Bonferroni correction and significance was defined as * P < 0.05; ** P < 0.01; *** P < 0.001 and **** P < 0.0001]

Article Snippet: Proteins were detected by immunoblotting using mouse anti‐human CD9 (clone HI9a, BioLegend, dilution 1:1000), mouse anti‐human CD63 (clone TS63, Abcam, dilution 1:1000), mouse anti‐human flotillin‐1 (clone 18, BD Biosciences, MA, USA, dilution 1:500, sample reduced with β‐mercaptoethanol) and mouse anti‐human HSC70/HSP70 (clone N27F3‐4, ENZO dilution 1:1000, sample reduced with β‐mercaptoethanol) and rabbit anti‐human lactoferrin (polyclonal, Abcam, dilution 1:5000), rabbit anti‐human TLR3 (D10F10, Cell Signaling TECHNOLOGY, dilution 1:1000, sample reduced with β‐mercaptoethanol), mouse anti‐human GAPDH (mAbcam 4984, Abcam, dilution 1:1000), mouse anti‐human CSTB (clone 225228, R&D Systems, dilution 1:500, sample reduced with β‐mercaptoethanol).

Techniques: Activation Assay, Cell Culture, Whisker Assay, Gene Expression, Expressing, Derivative Assay, Protein-Protein interactions, Western Blot, Purification, Control