Tissue staining for S1PR1 was performed using formalin-fixed, paraffin-embedded tissue sections mounted on glass slides. S1PR1 (Abcam, Cambridge, U.K.) and IgG1 isotypic control (R&D Systems, Abbingdon, U.K.) Abs were used at 4 μg/ml, whereas CD20 and CD23 were supplied ready to use (Dako, Cambridge, U.K.). As previously described (33 (link)), dewaxing of the sections and Ag retrieval were performed with EnVision FLEX target retrieval solution with the Dako PT-link module. Slides were stained with an autostainer using the EnVision FLEX convenience kit (Dako) and counterstained in Meyers’ hematoxylin (Sigma-Aldrich). Isotypic control staining is shown in Supplemental Fig. 2A .
S1pr1
Manufactured by Abcam
Sourced in United States, United Kingdom
S1PR1 is a membrane protein that functions as a receptor for sphingosine 1-phosphate (S1P). It plays a role in various cellular processes, including lymphocyte trafficking, vascular maturation, and neural function.
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Lab products found in correlation
Ero1α, by Santa Cruz Biotechnology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Envision flex target retrieval solution, by Agilent Technologies (1 mentions)
Envision flex convenience kit, by Agilent Technologies (1 mentions)
Meyer s hematoxylin, by Merck Group (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Erk1 2, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Occludin, by Abcam (1 mentions)
Tunel staining, by Roche (1 mentions)
P stat3, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Ripa buffer, by CWBIO (1 mentions)
Cleaved parp, by Abcam (1 mentions)
E cadherin, by R&D Systems (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
9 protocols using s1pr1
1
Immunohistochemical Staining for S1PR1
2
Immunohistochemical Analysis of Protein Expression
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Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. In citrate buffer (pH 6.0), slides were heated in an autoclave for 3 min for antigen retrieval. Slides were incubated with appropriate primary antibody (ERO1α from Santa Cruz; or S1PR1 from Abcam; or p-STAT3, VEGF-A, or CD34 from CST) overnight at 4 °C, followed by counterstaining with hematoxylin. The ERO1α expression status was graded by two independent observers as described by immunohistochemistry score: scoring was conducted based on the percentage of positive-staining cells: 0–5% scored 0, 6–35% scored 1, 36–70% scored 2, and more than 70% scored 3; staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2, and strongly staining scored 3. The final score was calculated using the percentage score × staining intensity score as follows: “0” for a score of 0–1, “1” for a score of 2–3, “2” for a score of 4–6, and “3” for a score of >6. In subsequent analyses, scores “0” and “1” were defined as the low group and scores “2” and “3” were defined as the high group scores.
3
Immunoblot Analysis of Tight Junction Proteins
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Samples containing equivalent amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5%-10%) and then were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 2 h in a blocking buffer [Tris-buffered saline with 0.05% Tween 20 (TBST) containing 5% skim milk] and then incubated overnight at 4 °C with primary antibodies (claudin-5, 1:1000, Invitrogen, Carlsbad, CA, USA; occludin, 1:1000, Abcam, Cambridge, MA, USA; S1PR1, 1:1000, Abcam, Cambridge, MA, USA; p-extracellular regulated protein kinases (ERK) 1/2, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA; ERK 1/2, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with corresponding secondary antibodies at room temperature for 1 h, the protein bands were estimated by Immobilon Western Chemiluminescent horseradish peroxidase substrate. All protein bands were visualized by enhanced chemiluminutesescence (ECL) kit (EMD Millipore, Billerica, MA, USA) and were recorded by Tanon 5500 Chemiluminescence Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). ImageJ software (US National Institutes of Health) was used to analyze the blots.
4
Immunohistochemical Profiling of Biomarkers
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Four micrometers specimen sections were deparaffinized and rehydrated; then antigen retrieval was performed. After that, endogenous peroxidase activity was blocked. After incubation of primary and secondary antibody, the slides were incubated with diaminobenzidine (DAB) and finally counterstained with hematoxylin. Primary antibodies are listed as follows: Ki-67 (1:500, Proteintech, USA), S1PR1 (1:200, Abcam, USA), p-STAT3 (1100, Abcam, USA), TUNEL staining (Roche, USA). The immunostaining intensity was classed into four categories: negative (value = 0), weak (value = 1), moderate (value = 2) and strong (value = 3). The percentage of positive cell was classed into four categories: 0–25% (value = 0), 26–50% (value = 1), 51–75% (value = 2) and 76–100% (value = 3).
5
Immunoblotting Protocol for Protein Analysis
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Immunoblotting was performed as a previous report (Zhang et al., 2021 (link)). Cells were lysed by using RIPA Buffer (CWBio, China) containing protease inhibitors to extract total protein. Total protein (20 μg) was added to 10% SDS-polyacrylamide gel. The protein was transferred to PVDF membranes (Millipore, USA) after electrophoresis and blocked for 1 hr with a blocking buffer (Beyotime, China). Then PVDF membranes were incubated with the primary antibodies including ALP (Abcam, UK, 1:3000), RUNX2 (CST, USA, 1:2,000), S1PR1 (Abcam, UK, 1:2,000), and GAPDH (CST, USA, 1:5,000) overnight at 4°C. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr and reacted with ECL (Millipore, USA). Finally, the photographs were taken by Tanon-5200 system (Tanon, China). The densitometry of protein bands was quantified by ImageJ 1.51K.
6
Comprehensive Antibody Analysis Protocol
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Antibodies used in the present study are as following: p21 (Abcam), pT145-p21 (Abcam), AKT1 (Santa Cruz), pS473-AKT1 (Santa Cruz), Vimentin (Abcam), β-catenin (Abcam), GAPDH (Abcam), β-actin (Sigma), cleaved PARP (Abcam), active caspase-3 (Cell signaling), procaspase-3 (Abcam), Oct4 (Abcam), E-cadherin (R & D), S1PR1 (Abcam).
7
Analysis of Sphingosine Kinase and S1P Receptor Signaling
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Twenty-five milligrams of detrusor tissue was ground on ice and added to Ripa lysate buffer containing phenylmethane sulfonylfluoride and a phosphatase inhibitor. The protein concentration was measured using a bicinchoninic acid protein detection kit (Beyotime Biotechnology, China). The protein samples were separated by electrophoresis in 10% twelve alkyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride film. The membrane was sealed in Tris-Buffered Saline Tween-20 with 5% skim milk for 1 h and incubated with the primary antibody at 4°C overnight. The solution was replaced the next day with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000) (Boster Biological Technology, China) and incubated at room temperature for 1 h. After washing, the band was visualized with a super-sensitive electrogenerated chemiluminescence (ECL) chemiluminescence solution (Haigene Detection Co. Ltd, China) and analyzed with a Chemidoc MP chemiluminescence imaging system (Bio-Rad, USA). Actin was used as the internal reference and the following proteins were examined: Actin (Abcam, UK), SphK1 (Abcam), SphK2 (Thermo, USA), S1PR1 (Abcam), S1PR2 (Thermo), S1PR3 (Abcam), RhoA (Abcam), ROCK1 (Cst, USA), ROCK2 (Cst), MYPT1 (Cst), p-MYPT1 (Cst), MLC20 (Cst), and p-MLC20 (Cst).
8
Quantitative Western Blot Analysis of Signaling Proteins
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Concentrations of proteins extracted from cell lines or tumor tissues were determined by bicinchoninic acid assay kits (Beyotime, Shanghai, China) following the manufacturer’s instructions. Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% skim milk powder at room temperature for 2 h, membranes were incubated with primary antibody (1:1000) overnight at 4 °C. Appropriate secondary antibody (1:2000) was added before incubation for 2 h at 37 °C. Protein band intensities were measured using Image Lab software. GAPDH was used as the internal control. Western blots were quantified using Image Pro Plus version 6. Primary antibodies were from rabbits against S1PR1, VEGF-A (Abcam, Cambridge, UK), STAT3, p-STAT3, E-cadherin, vimentin, Slug, and GAPDH (Cell Signaling Technology, Danvers, MA, USA); and from mice against ERO1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
9
Western Blot Analysis of Signaling Proteins
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Tissue samples were homogenized in protein lysis buffer and the samples were centrifuged at 12000 ×g for 10 min. The supernatant was collected and used for Western blotting. For the in vitro study, cancer cells were plated in 60 mm dishes and treated with the drug for 24 h. Cells were lysed in protein lysis buffer and used for further analysis. Proteins were separated on polyacrylamide gels and transferred to nitrocellulose membranes. After the transfer, membranes were blocked with 5% skimmed milk and subsequently incubated with either of the following primary antibodies; S1PR1 (ab23695, 1:3000) was obtained from Abcam. STAT3 (sc-482, 1:2000), c-MYC (sc-764, 1:3000), E-Cadherin (sc-7870, 1:1000), N-Cadherin (sc-7939, 1:2000), Cyclin-D1 (sc-753, 1:1000), COX-2 (sc-7951, 1:1000), ERK 1 (sc-93, 1:3000), and β-Tubulin (sc-9104, 1:2000) were procured from Santa Cruz Biotechnology. p-STAT3 (9145S, 1:1000), Vimentin (5741, 1:3000), p-ERK 1/2 (9106, 1:2000), and p-Akt (9271, 1:1000) were purchased from cell signaling Technology. HRP conjugated secondary antibody was added and the detection were performed using ECL solution.
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