The AGS, MKN45, HGC27, SNU1, KATOIII, Hela and HEK293T cells were obtained from the American Type Culture Collection (ATCC). Human GCa cell lines MGC803 were purchased from China Academia Sinica (Shanghai, PR China). The GCa MGC803, AGS, MKN45, HGC27, SNU1, KATOIII cells were cultured in RPMI-1640 medium and Hela cells, HEK293T embryonic kidney cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO2. Antibodies against the following proteins were used with source and dilution ratios indicated: ATG4B (Cell signaling, #13507, 1:1000 and Proteintech, # Cat No. 15131-1-AP, 1:1000); P62 (Sigma, #P0067, 1:1000); LC3 (Sigma, #ABC929, 1:1000; immunofluorescence 1:100); LAMP1(CST, #15665S, 1:100); Snail (CST, #4719, 1:1000); N-cadherin (CST, #4061, 1:1000); C-caspase7 (CST, #12827, 1:1000); PARP-1 (CST, #9542, 1:1000); GAPDH (CST, #2118, 1:1000); Anti-rabbit IgG Fab2 (CST, #4412s, 1:500); Anti-mouse IgG Fab2 (CST, #4409s, 1:500). AzalomycinF4a was isolated from Streptomyces solisilvae HNM30702 and verified by the NMR and HRESIMS data [34 (link)]. Rapamycin (MCE, #HY-10219); BafilomycinA1 (MCE, # HY-100558); Tioconazole (MCE, #HY-1303191 CS-2360); Acridine Orange (Sigma, #MKCD9806); FMK9a (MCE, HY-100522); DAPI (Beyotime, ON.C1005).
Snail
Manufactured by Merck Group
Sourced in United States
Snail is a compact and versatile laboratory equipment designed for various scientific applications. It serves as a platform for the observation and study of small organisms, such as snails, in a controlled environment. The core function of Snail is to provide a secure and stable setting for the examination and experimentation of these specimens.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Merck Group (5 mentions)
E cadherin, by Merck Group (5 mentions)
Gapdh, by Merck Group (4 mentions)
β catenin, by Merck Group (4 mentions)
N cadherin, by Merck Group (3 mentions)
E cadherin, by Abcam (2 mentions)
Parp1, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Lamp1, by Merck Group (1 mentions)
Atg4b, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Hgc 27, by American Type Culture Collection (1 mentions)
Mkn45, by American Type Culture Collection (1 mentions)
Snu 1, by American Type Culture Collection (1 mentions)
Kato 3, by American Type Culture Collection (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Transwell chamber, by Merck Group (1 mentions)
Gapdh, by Vazyme (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
11 protocols using snail
1
Anticancer Effects of AzalomycinF4a
2
Ovarian Cancer Stem Cell Protocol
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Human ovarian cancer stem cells were screened and collected at Central Laboratory of Shandong University School of Medicine (Jinan, China). The pcDNA3.1-WWOX eukaryotic expression vector was also prepared and stored in this laboratory. The LipofectorTM liposomal transfection reagent was provided by the Beyotime Institute of Biotechnology (Shanghai, China) and Transwell® chambers were purchased from Chemicon (Billerica, MA, USA). The E-cadherin, β-catenin, vimentin, fibronectin, Elf5 and Snail primary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). This study was approved by the Ethics Committee of Shandong University (Jinan, China).
3
Quantification of MFAP2 Expression
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Total RNA was extracted from cell samples using TRIzol reagent in accordance with the instructions of the manufacturer (Invitrogen, Carlsbad, CA, USA). We used the cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) to synthesize first-strand cDNA. Moreover, RT-PCR was carried out with the use of SYBR Green PCR Master Mix (Applied Biosystems) to detect the transcript abundance of MFAP2. The following primer pairs were used for MFAP2:
The calculation of the relative quantification was performed according to the method of 2−ΔΔCt.
Western blot analysis. For the detection of the protein expression, Western blot analysis was carried out as previously described. The antibodies used were: MFAP2 (Abcam, UK), E-cadherin (Sigma-Aldrich, Chicago, IL, USA), vimentin (Sigma-Aldrich, Chicago, IL, USA), Snail (Sigma-Aldrich, Chicago, IL, USA), β-catenin (Sigma-Aldrich, Chicago, IL, USA), and GAPDH (Vazyme, Piscataway, NJ, USA), using standard chemical luminescence methodology.
The calculation of the relative quantification was performed according to the method of 2−ΔΔCt.
Western blot analysis. For the detection of the protein expression, Western blot analysis was carried out as previously described. The antibodies used were: MFAP2 (Abcam, UK), E-cadherin (Sigma-Aldrich, Chicago, IL, USA), vimentin (Sigma-Aldrich, Chicago, IL, USA), Snail (Sigma-Aldrich, Chicago, IL, USA), β-catenin (Sigma-Aldrich, Chicago, IL, USA), and GAPDH (Vazyme, Piscataway, NJ, USA), using standard chemical luminescence methodology.
4
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Proteins from cell and tissue lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corp, Port Washington, NY). Then the membranes were blocked with 5% skimmed milk and incubated using primary antibodies against IMPDH2 (1:1000 dilution), anti-GAPDH, anti-GSK3β, anti-p-GSK3β, anti-AKT, anti-p-AKT (Ser473), anti-FOXO1, anti-p-FOXO1, anti-mTOR, anti-p-mTOR (Cell signaling Technology, Beverly, MA), E-cadherin (1:1000 dilution,#SAB4503751; Sigma Aldrich), β-catenin (1:1000 dilution, #C2206; Sigma Aldrich), Vimentin (1:1000 dilution, #V6630; Sigma Aldrich), Snail (1:1000 dilution, #SAB1306281; Sigma Aldrich), followed by incubation with the appropriate secondary antibodies (anti-rabbit IgG, 1:3000 dilution, #7074; Cell Signaling). An enhanced chemiluminescence (Pierce, Rockford, IL, USA) was used to detect signals.
5
Quantifying Lung Tissue Protein Expression
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The expression levels of Carbonic anhydrase IX (CAIX), HDAC3, Snail, and FOXA1 were evaluated by immunohistochemistry. Sections with a 6-μm thickness were prepared by slicing lung tissue blocks embedded in paraffin. Slides were deparaffinized in xylene and rehydrated through a series of alcohols in water. Antigen retrieval was performed by incubation in citrate buffer (pH 6) in a microwave for 20 min. Endogenous peroxidase activity was blocked by adding 3% H2O2 for 10 min. After the samples were incubated overnight with primary antibodies against CAIX (Novus), HDAC3 (GeneTex, Irvine, CA, USA), Snail (Sigma–Aldrich), and FOXA1 (Abcam) at 4 °C, the samples were rinsed and incubated with secondary antibodies. Diaminobenzidine (Roche, Germany) was added, and the slides were incubated in the dark for 10 min. Expression levels were quantified using ImageJ software.
6
Investigating FAK Signaling Pathway
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Antibodies specific for the following proteins and tags were used: FAK (Abcam, #ab76496), ZDHHC5 (CST, #79842), β-actin (Proteintech, #81115–1-RR), Ubiquitin (CST, #20326), Flag (Abcam, #ab205606), Myc (Abcam, #ab9106), HA (CST, #3724), phospho-FAK (Abcam, #ab81298), Paxillin (Abcam, #ab32084), phospho-Paxillin (Abcam, #ab109547), AKT (Abcam, #ab8805), phospho-AKT (Abcam, #ab81283), ERK (Abcam, #ab184699), phospho-ERK (Abcam, #ab201015), α-Tubulin (Proteintech, #11,224–1-AP), ATP1A1 (Abcam, #ab7671), E-cadherin (Abcam, #ab219332), N-cadherin (Abcam, #ab18203), snail (Sigma-Aldrich, #SAB5700703), slug (Sigma-Aldrich, #SAB5700672), and β-catenin (Abcam, #ab32572).
The following reagents were used: MG132 (Beyotime, #S1748), cycloheximide (Sigma-Aldrich, #66–81-9), 2-bromopalmitate (Sigma-Aldrich, #238442), palmostatin B (Sigma-Aldrich, #178501), DMSO (Beyotime, #ST038), MeO-PEG-Mal (Sigma-Aldrich, #63187), hydroxylamine (Sigma-Aldrich, #159417), N-ethylmaleimide (NEM) (Sigma-Aldrich, #04260), and TCEP (Sigma-Aldrich, #646547).
The following reagents were used: MG132 (Beyotime, #S1748), cycloheximide (Sigma-Aldrich, #66–81-9), 2-bromopalmitate (Sigma-Aldrich, #238442), palmostatin B (Sigma-Aldrich, #178501), DMSO (Beyotime, #ST038), MeO-PEG-Mal (Sigma-Aldrich, #63187), hydroxylamine (Sigma-Aldrich, #159417), N-ethylmaleimide (NEM) (Sigma-Aldrich, #04260), and TCEP (Sigma-Aldrich, #646547).
7
Protein Expression Analysis by Western Blot
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Western blot assay was performed referring to several previously published works [24–26 (link)]. Briefly, proteins were extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad, Hercules, CA, USA). The separated proteins were subsequently electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), which were then soaked in 5% nonfat milk for a while. The membranes were then incubated with primary antibodies against ZNF267 (HPA003866, Sigma-Aldrich, Darmstadt, Germany), E-cadherin (SAB4503751), N-cadherin (SAB5700641), snail (SAB1306281), CD44 (SAB4300691), CD133 (SAB4300882), OCT4 (SAB5100006), and GAPDH (G9545, Sigma-Aldrich), followed by another one hour of incubation with secondaries (A0545, 1:10,000, Sigma-Aldrich). Finally, the blotting was enhanced using ECL reagents (Pierce, Rockford, IL, USA).
8
Protein Expression Analysis in Glioblastoma
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Protein extraction and Western blot analysis were performed as previously described.18 (link) Representative images from 2 or 3 independent experiments are shown. The antibodies that were used included c-MET, E-cadherin, N-cadherin, Vimentin, ZEB1, Slug, Snail and GAPDH (Sigma Aldrich).
To detect c-MET expression in glioblastoma, IHC staining was performed on tumor tissue using methods described previously.17 (link) c-MET were scored by an IHC score based on staining intensity and percentage of positive cells within the whole tissue section.
To detect c-MET expression in glioblastoma, IHC staining was performed on tumor tissue using methods described previously.17 (link) c-MET were scored by an IHC score based on staining intensity and percentage of positive cells within the whole tissue section.
9
Western Blotting for Protein Expression
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Western blotting was adopted during the process to determine the degrees of protein expression. The antibodies included are DUSP14 (cat. no. ab134265, 1:800, Abcam, Cambridge, UK), E-cadherin (cat. no. SAB4503751, 1:500, Sigma-Aldrich, Chicago, IL, USA); Vimentin (cat. no. SAB1305433, 1:1000, Sigma-Aldrich, Chicago, IL, USA); Snail (cat. no. SAB4502825, 1:800, Sigma-Aldrich, Chicago, IL, USA); β-catenin (cat. no. SAB4500541, 1:800, Sigma-Aldrich, Chicago, IL, USA) and/or GAPDH (cat. no. 10494-1-AP, 1:5000, Proteintech Group, Chicago, IL, USA). Signals were detected in accordance with the standard methodologies for chemoluminescence.
10
Quantitative Western Blot Analysis
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After incubation with indicated factors cells were harvested in lysis buffer (0.0625 M Tris/HCl pH: 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol) and total protein concentration was evaluated according to Pedersen protocol. Lysates containing equal amounts of protein were separated in 10% SDS-PAGE gel and transferred onto PVDF membrane. For protein detection specific primary antibodies against: AR, p-AR(Ser210/213), E-cad, p-Ecad(Ser838/Ser840), AKT, p-AKT(Ser 473), PTEN, p-PTEN(Ser380), RAS, RAF, ERK1/2, BAX, BAD, BCL-2, SNAIL, TWIST, ZEB1 and β-actin (Sigma-Aldrich, Poland) as for referral protein were used. Secondary rabbit or mouse antibodies conjugated with HRP (1:10,000, Cell Signaling Technology, Inc.) were utilised for detection. All steps were performed as previously described [22] . In order to obtain quantitative results, the bands (representing each data point) were densitometrically scanned using SynGene Gene Tools version 4.03.0 (Synoptics Ltd Beacon House, Nuffield Road Cambridge, CB4 1TF, UK). β-actin was used as a control. Protein level within the control group was arbitrarily set as 1. The procedure was performed as previously described [24] (link).
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