Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Zuo Jin Wan Reverses the Resistance of Colorectal Cancer to Oxaliplatin by Regulating the MALAT1/miR-200s/JNK Signaling Pathway

doi: 10.1155/2022/3032407

Figure Lengend Snippet: Primer sequences.

Article Snippet: The primary antibodies used were MDR1/ABCB1 (CST, USA), ABCG2 (CST, USA), MRP4/ABCC4 (CST, USA), JNK2 (CST, USA), phospho-SAPK/JNK (CST, USA), and GAPDH (CST, USA).

Techniques: Sequencing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Zuo Jin Wan Reverses the Resistance of Colorectal Cancer to Oxaliplatin by Regulating the MALAT1/miR-200s/JNK Signaling Pathway

doi: 10.1155/2022/3032407

Figure Lengend Snippet: Effect of ZJW on the expression of the drug resistance-related proteins MDR1/ABCB1, MRP4/ABCC4, and ABCG2 in HCT116/L-OHP cells. (a and b) Western blot assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 proteins in HCT116 cells and HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. (c) Real-time PCR assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 levels in HCT116 and HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. ∗ , P < 0.05; ∗∗ , P < 0.01; ∗∗∗ , P < 0.001 compared to HCT116/L.

Article Snippet: The primary antibodies used were MDR1/ABCB1 (CST, USA), ABCG2 (CST, USA), MRP4/ABCC4 (CST, USA), JNK2 (CST, USA), phospho-SAPK/JNK (CST, USA), and GAPDH (CST, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Zuo Jin Wan Reverses the Resistance of Colorectal Cancer to Oxaliplatin by Regulating the MALAT1/miR-200s/JNK Signaling Pathway

doi: 10.1155/2022/3032407

Figure Lengend Snippet: Effect of ZJW on the expression of drug resistance-related proteins in vivo. (a and b) Immunofluorescence detection of MDR1/ABCB1 and ABCG2 proteins in vivo. (c and d) Immunohistochemistry detection of MRP4/ABCC4 protein in vivo. (e and f) Western blot assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 levels in vivo. (g) Real-time PCR assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 levels in vivo. ∗ , P < 0.05; ∗∗ , P < 0.01; ∗∗∗ , P < 0.001; ∗∗∗∗ , P < 0.0001; ns, no significance compared to MODEL.

Article Snippet: The primary antibodies used were MDR1/ABCB1 (CST, USA), ABCG2 (CST, USA), MRP4/ABCC4 (CST, USA), JNK2 (CST, USA), phospho-SAPK/JNK (CST, USA), and GAPDH (CST, USA).

Techniques: Expressing, In Vivo, Immunofluorescence, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

doi: 10.2147/IJN.S107952

Figure Lengend Snippet: In vitro cytotoxicity studies of LR, PSLR, and EPSLR. Notes: Studies of LR, PSLR, and EPSLR for 24 ( A ), 48 ( B ), and 72 hours ( C ) ( P <0.05) and Western blot. Quantification of Western blot bands using ImageJ ( D ). MDR1 silencing mediated by control, naked siRNA, LR NC , LR, PSLR, and EPSLR ( E ). Abbreviations: LR, liposome–siRNA complexes; siRNA, small interfering RNA; PSLR, PEGylated LR; EPSLR, PSLR-conjugated anti-EphA10 antibody; LR NC , Liposome–negative control siRNA complexes; N/P ratios, molar ratio of DOTAP-nitrogen atoms to siRNA-phosphate; P-gp, P-glycoprotein.

Article Snippet: MDR1 and β-actin protein were detected using the MDR1/ABCB1 rabbit monoclonal antibody (1:300; Cell Signal Technology) and the rabbit anti-β-actin antibody at a dilution of 1:1,000, respectively.

Techniques: In Vitro, Western Blot, Small Interfering RNA, Negative Control

Journal: BMC Cancer

Article Title: Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

doi: 10.1186/1471-2407-13-93

Figure Lengend Snippet: Paclitaxel resistant gene candidates in pooled samples. A ) Candidate ‘hits’ identified in resistant pools of four cell lines. Genes found in multiple cell lines are color-coded and labeled. Dot surfaces and numbers within parentheses indicate insertion occurrence, and y-axis indicates total read numbers for each gene. B ) Venn diagram showing candidate genes belonging to four cell lines. Only one gene (ABCB1) was shared by all four cell lines. C ) Functional annotation analysis of pooled samples. Only genes with multiple hits were used in DAVID analysis. Only annotation groups with significant values (p-value<0.05, FDR<5%) are listed. The complete annotation chart and cluster chart are presented in the Additional file Dataset S4.

Article Snippet: The membrane was blotted with MDR1/ABCB1 rabbit polyclonal antibody (Cell Signaling Technology #12273) diluted by 2,500-fold, and goat anti-rabbit IgG (Thermo Scientific #31460) diluted by 10,000-fold.

Techniques: Labeling, Functional Assay

Journal: BMC Cancer

Article Title: Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

doi: 10.1186/1471-2407-13-93

Figure Lengend Snippet: ABCB1 as the primary resistant gene. A ) Insertion sites near ABCB1 genomic locus are enriched in resistant samples. Insertion sites of a prescreened library and resistant pools in Chr7:85000000–89500000 are plotted. Scale is drawn as per Mb. Dot surfaces indicate number of positive samples, and y-axis indicates normalized read numbers as a percentage of total signals. Read numbers are unfiltered. All annotated genes within this region are shown as arrows. A blow-up view indicates ABCB1 genomic arrangement with open reading frame shown as yellow boxes. Asterisk denotes exon 3 with the ATG start codon (chr7:87229506). Insertion sites confirmed by TOPO cloning and Sanger sequencing are drawn as triangles above the diagram with direction of arrows indicating orientation of the CMV and the splice donor. HP, MP, TP1-3 denote HeLa, MCF7, and T47D paclitaxel resistant clones respectively. Colors of dots indicate orientation of the CMV with forward orientation relative to ABCB1 as blue and reverse orientation as yellow. B ) PB insertions activate ABCB1 expression. Error bars show standard deviation (n=3). Significances are indicated by p-values. “pre” denotes prescreened libraries; MP, HP, and TP1-3 denote clones shown above. C ) Detection of the chimeric mRNA in clones with insertions in the ABCB1 intron. Top panel (PB/ABCB1-E3) shows 404bp chimeric PCR products with a transposon-specific primer and an ABCB1 exon 3 primer. A lower band at 300bp could be due to alternative splicing. Bottom panel (hPBGD) shows the 151bp PCR products using the primer pair amplifying the porphobilinogen deaminase (PBGD) housekeeping gene. Three native cell lines (H, HeLa; M, MCF7; T, T47D) and a HeLa clone with PB insertions but not in the ABCB1 gene (HN) were used as controls. The first lane (MK) indicates 100bp DNA ladder (New England Biolabs).

Article Snippet: The membrane was blotted with MDR1/ABCB1 rabbit polyclonal antibody (Cell Signaling Technology #12273) diluted by 2,500-fold, and goat anti-rabbit IgG (Thermo Scientific #31460) diluted by 10,000-fold.

Techniques: Clone Assay, Sequencing, Expressing, Standard Deviation

Journal: BMC Cancer

Article Title: Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

doi: 10.1186/1471-2407-13-93

Figure Lengend Snippet: Candidate hits in resistant clones. A ) Cluster analysis of IMR32 resistant clones. X-axis indicates colonies and y-axis indicates insertion sites. Colonies within a cluster have same insertions and are likely derived from one founder clone. Insertions are in either same (red) or opposite (blue) orientations of a gene. B ) Paclitaxel sensitivity curve of IMR32 transfected with a control (CTL) or ABCB1 cDNA plasmid (ABCB1). Cell survival was measured by CellTiter-Glo assay. C ) Western blot showing ABCB1 overexpression in IMR32 cells transfected with pCMV-ABCB1 plasmid. CTL, cells transfected with a control pCMV plasmid; ABCB1, cells transfected with pCMV-ABCB1 cDNA plasmid. ABCB1 was shown as bands at 150kD. Actin was used as loading control. D ) PB insertions in MEIS1 gene. The direction of gene is drawn from left to right, with yellow squares indicating exons. Forward strand insertion sites are drawn as green triangles and reverse as red triangles. Scale is drawn as per kb. E ) Paclitaxel sensitivity profile in a panel of cancer cell lines. Cell lines are either divided to two groups by median ABCB1 or MEIS1 mRNA levels, n=143, (first and second graphs), or first divided by median ABCB1 levels and then by median MEIS1 mRNA levels, n=72 (ABCB1 Low) and 71 (ABCB1 High). Red bars indicate median IC50.

Article Snippet: The membrane was blotted with MDR1/ABCB1 rabbit polyclonal antibody (Cell Signaling Technology #12273) diluted by 2,500-fold, and goat anti-rabbit IgG (Thermo Scientific #31460) diluted by 10,000-fold.

Techniques: Clone Assay, Derivative Assay, Transfection, Plasmid Preparation, Glo Assay, Western Blot, Over Expression

Journal: BioMed Research International

Article Title: GDF15 Repression Contributes to 5-Fluorouracil Resistance in Human Colon Cancer by Regulating Epithelial-Mesenchymal Transition and Apoptosis

doi: 10.1155/2020/2826010

Figure Lengend Snippet: The low expression of GDF15 in 5-FU-resistant cells was associated with enhanced migration and antiapoptotic ability. (a) Western blotting analysis of MDR1, MRP1, MMP14, N-cadherin, E-cadherin, Bcl-2, cleaved caspase-3, and Bax levels in HCT-15 and HCT-15/FU cells treated with 0.76 mM 5-FU for 48 hours. (b) GDF15 mRNA and protein level in HCT-15 and HCT-15/FU cells treated with 0.76 mM 5-FU for 48 h. Upper: the relative mRNA expression of GDF15 in HCT-15 and HCT-15/FU cells; lower: western blotting analysis of GDF15 and the downstream signaling pathway protein P-Smad2/3 in HCT-15 and HCT-15/FU cells. The data represent the mean ± SEM. Statistical significance: ∗∗∗ P < 0.001.

Article Snippet: The specific primary antibodies used in western blot analysis were as follows: anti-MDR1 (1 : 1000, 13342, CST), anti-MRP1 (1 : 1000, 72202S, CST), anti-E-cadherin (1 : 5000, Cat. No. 20874-1-AP, Proteintech), anti-N-cadherin (1 : 5000, ab76011, Abcam), anti-BAX (1 : 1000, ab32503, Abcam), anti-Bcl-2 (1 : 2000, ab182858, Abcam), anti-cleaved caspase-3 (1 : 1000, 9664S, CST), anti-MMP14 (1 : 2000, ab51074, Abcam), anti-p-Smad2/3 (1 : 500, Hangzhou HuaAn Biotechnology), anti-Smad2/3 (1 : 1000, Hangzhou HuaAn Biotechnology), and anti-GAPDH (1 : 5000, Cat. No. 60004-1-Ig, Proteintech).

Techniques: Expressing, Migration, Western Blot

Journal: BioMed Research International

Article Title: GDF15 Repression Contributes to 5-Fluorouracil Resistance in Human Colon Cancer by Regulating Epithelial-Mesenchymal Transition and Apoptosis

doi: 10.1155/2020/2826010

Figure Lengend Snippet: The overexpression of GDF15 resensitized the 5-FU-resistant HCT-15/FU cells to 5-FU. (a) The mRNA expression of GDF15 in HCT-15/FU cells treated with 0.76 mM 5-FU for 0 h or 48 h after being transfected with pCMV-GDF15 or empty vector. (b) Western blotting analysis of GDF15 in HCT-15/FU cells treated with 0.76 mM 5-FU for 0 h or 48 h after being transfected with pCMV-GDF15 or empty vector. (c) The MTS assay to detect inhibition of growth by 5-FU in HCT-15/FU cells treated with 5-FU after being transfected with pCMV-GDF15 or empty vector. (d, e) The cell migration assay of HCT-15/FU cells treated with 0.76 mM 5-FU for 0 h or 48 h after being transfected with pCMV-GDF15 or empty vector. (d) The transwell migration assay (scale bars, 50 μ m) and (e) wound healing assay (scale bars, 200 μ m). (f) The flow cytometric apoptosis assays of HCT-15/FU cells treated with 0.76 mM 5-FU for 0 h or 48 h after being transfected with pCMV-GDF15 or empty vector. (g) Western blotting analysis of MDR1, MRP1, MMP14, N-cadherin, E-cadherin, Bcl-2, cleaved caspase-3, and Bax levels in HCT-15/FU cells treated with 0.76 mM 5-FU for 48 h after being transfected with pCMV-GDF15 or empty vector. The data represent the mean ± SEM. Statistical significance: ∗∗∗ P < 0.001.

Article Snippet: The specific primary antibodies used in western blot analysis were as follows: anti-MDR1 (1 : 1000, 13342, CST), anti-MRP1 (1 : 1000, 72202S, CST), anti-E-cadherin (1 : 5000, Cat. No. 20874-1-AP, Proteintech), anti-N-cadherin (1 : 5000, ab76011, Abcam), anti-BAX (1 : 1000, ab32503, Abcam), anti-Bcl-2 (1 : 2000, ab182858, Abcam), anti-cleaved caspase-3 (1 : 1000, 9664S, CST), anti-MMP14 (1 : 2000, ab51074, Abcam), anti-p-Smad2/3 (1 : 500, Hangzhou HuaAn Biotechnology), anti-Smad2/3 (1 : 1000, Hangzhou HuaAn Biotechnology), and anti-GAPDH (1 : 5000, Cat. No. 60004-1-Ig, Proteintech).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, MTS Assay, Inhibition, Cell Migration Assay, Transwell Migration Assay, Wound Healing Assay

Journal: International Journal of Biological Sciences

Article Title: Discovery and Validation of Nitroxoline as a Novel STAT3 Inhibitor in Drug-resistant Urothelial Bladder Cancer

doi: 10.7150/ijbs.63125

Figure Lengend Snippet: Proposed mechanism for NTX-induced P-gp reversal, G0/G1 arrest and apoptosis in drug-resistant T24 cells. The molecular structure of STAT3 is classic in STAT family, consisting of N-term Domain for cooperative DNA binding, Coiled-coil Domain for STAT3 recruitment to a receptor, DNA binding Domain, Linker Domain, SH2 Domain for STAT3 dimerization, and Transactivation Domain for transcription activation. (B) STAT3 can be activated to signal through both canonical and non-canonical pathways. For the canonical pathway, NTX inhibits STAT3 phosphorylation at Y705 residue and then decreases the translocation of STAT3 dimers to the nucleus in T24/DOX and T24/CIS cells. The subsequent downregulation of target genes including MDR1 gene, the cell cycle regulatory genes such as c-Myc, Cyclin D1, and the anti-apoptotic genes such as Survivin, Mcl-1, Bcl-xL, will reverse P-gp, trigger G0/G1 arrest, and induce apoptosis, respectively. For the non-canonical pathway, NTX-downregulated STAT3 phosphorylation at S727 residue reduces the translocation of p-STAT3 (S727) to the mitochondria and finally induces cell apoptosis by the increased generation of reactive oxygen species (ROS).

Article Snippet: Primary antibodies against β-Actin (#4970), STAT3 (#12640), p-STAT3 (Y705) (#9145), p-STAT3 (S727) (#9134), MDR1/P-gp (#13342), c-Myc (#5605), Cyclin D1 (#2978), CDK4 (#2906), CDK6 (#3136), Bcl-xL (#2764), Mcl-1 (#4572), and Survivin (#2808) were provided by Cell Signaling Technology (CST, USA).

Techniques: Binding Assay, Activation Assay, Translocation Assay

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: Comparison of MDR1, MMP-9, and BAX and gray value of two groups of cells. (a) Protein bands. (b) Statistical graphs of protein bands. ∗ P < 0.05 and ∗∗ P < 0.01, DDP STAT3(-) group vs. negative control group. Note: A is a DDP STAT3(-) group, and B is a negative control group.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Negative Control

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: STAT3 expression and statistical results in gastric cancer and adjacent tissues. (a) Protein bands. (b) Statistical graphs of protein bands. ∗∗ P < 0.01, cancer tissue group vs. adjacent tissue group. Note: 1 is cancer tissue, and 2 is adjacent tissue.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Expressing

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: STAT3 expression in gastric cancer and adjacent tissues (40x, 100x, 200x, and 400x).

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Expressing

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: The statistics after STAT3 being knocked out. (a) Protein bands. (b) Statistical graphs of protein bands. ∗∗ P < 0.01, STAT3 knockout group vs. negative control group. Note: A is the STAT3 knockout group, and B is the negative control group.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Knock-Out, Negative Control

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: OD values of the two groups of cells at different time points. ∗ P < 0.05 and ∗∗ P < 001, DDP STAT3(-) group vs. DDP group.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques:

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: Comparison of MDR1, MMP-9, and BAX and gray value of two groups of cells. (a) Protein bands. (b) Statistical graphs of protein bands. ∗ P < 0.05 and ∗∗ P < 0.01, DDP STAT3(-) group vs. negative control group. Note: A is a DDP STAT3(-) group, and B is a negative control group.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Negative Control

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: Apoptosis of each group. (a–i) Flow cytometry apoptotic quadrant diagram. (j) Statistical graph of apoptosis rate. ∗ P < 0.05, DDP STAT3(-) group vs. negative control group. Note: A means negative control; B means FITC negative control; C means PI negative control; D,E, and F means the DDP group, G,H, and I was the DDP STAT3(-) group. Q1: annexin V-FITC-/PI+; the cells in this area are necrotic cells. There may also be a few late apoptotic cells; Q2: annexin V + FITC+/PI+; the cells in this area were late apoptotic cells; Q3: annexin V-FITC+/PI-; the cells in this area were early apoptotic cells; Q4: annexin V-FITC-/PI−; the cells in this area were living cells.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Flow Cytometry, Negative Control

Journal: Contrast Media & Molecular Imaging

Article Title: Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells

doi: 10.1155/2022/1426343

Figure Lengend Snippet: Two groups of cell growth cycles of two groups. (a, b) Schematic of the cell cycle. (c) Cell cycle statistics. ∗∗ P < 0.01, DDP STAT3(-) group vs. negative control group.

Article Snippet: The reagents and kits used in the present study were purchased as follows: cisplatin-resistant human gastric cancer cells (DDP) were purchased from Beina Bio (BNCC342230), DH5a competent Escherichia coli (c1100), ampicillin (A7490), puromycin (P8230) LB medium (L1010), T4 ligase (T1410), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (CA1020) were purchased from Beijing Soleibao Technology Co., Ltd. Fetal Bovine Serum (Gibco, 10099-141, Australia), CCK8 (Tongren, Japan / Toyohito, Japan, CK04), Trypsin (Gibco, R001100, Shanghai First Biochemical Pharmaceutical Co., Ltd.), STAT3 Antibody (9139, Osaka, Japan), MDR1 Antibody (CST, 13342, Shanghai Ltd.), BAX antibody MMP-9 (CST,13667, Abcam China), β-actin polyclonal antibody (APPLY GEN, C1828, AmyJet Scientific Inc, China).

Techniques: Negative Control

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Transcriptomic profiling identified differentially regulated miRNAs, EMTs, and drug resistance genes in Rb tumor subtypes. ( A ) Volcano plot showing differentially regulated miRNAs in Rb subjects ( n = 9) compared with the pediatric retinas ( n = 2) identified using a microarray. ( B ) Heatmap showing differential expression of miRNAs in 9 Rb subjects and 2 pediatric controls identified using a microarray. ( C ) Bubble scatter plot showing the top enriched KEGG pathways regulated by miRNAs in Rb tumors. ( D ) RT-PCR results showing the normalized expression of miR-181a-5p in the control retinas ( n = 4), advanced Rb ( n = 4), and non-advanced Rb ( n = 4). Volcano plot showing the differentially regulated EMT and chemotherapy resistance genes identified using a microarray in ( E ) advanced Rb tumors and ( F ) non-advanced Rb tumors. ( G ) Heatmap showing the expression of EMT and chemotherapy resistance genes in 9 Rb subjects and 2 pediatric controls. RT-PCR showing the normalized expression of ( H ) ZEB1 , ( I ) SNAI2 , ( J ) ABCB1 , and ( K ) CTSL in control pediatric retinas ( n = 4), advanced Rb tumors ( n = 4), and non-advanced Rb tumors ( n = 4). Values represent the mean ± s.d. Two-tailed Mann–Whitney was used for the statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. ’ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The cell surface expression of MDR1 in parental and resistant Y79 and WERI-Rb1 cells was detected using an anti-MDR1 antibody (1:500, cat#13342, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Validation of epithelial-to-mesenchymal transition (EMT) and chemo-drug resistance genes in Rb tumors and their correlation with miR-181a-5p. Immunofluorescence results showing the expression of ( A ) ZEB1 and CTSL. The IF mean intensity of ( B ) ZEB1 and ( C ) cathepsin L staining in advanced tumors ( n = 12), non-advanced tumors ( n = 12), and control pediatric retinas ( n = 4). Immunofluorescence results showing the expression of ( D ) N-cadherin and E-cadherin, IF mean intensity of ( E ) N-cadherin and ( F ) E-cadherin in advanced tumors ( n = 12), non-advanced tumors ( n = 12), and control pediatric retina tissues ( n = 4). Scale bar: 50 µm. ( G ) Network map showing the predicted interaction of miRNA–mRNA targets using miRNet. Correlation plot showing the ( H ) negative correlation of EMT genes ( ZEB1, SNAI2, and TWIST ) with miR-181a-5p in Rb tumors and ( I ) negative correlation of drug resistance genes ( MDR1, MRP1 , and CTSL ) with miR-181a-5p in Rb tumors. Values represent the mean ± s.d. Two-tailed Mann–Whitney was used for statistical analysis. ** p < 0.01, *** p < 0.001. ’ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The cell surface expression of MDR1 in parental and resistant Y79 and WERI-Rb1 cells was detected using an anti-MDR1 antibody (1:500, cat#13342, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Immunofluorescence, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Chemotherapy-resistant Rb cells conferred a high-EMT program and metastasis. ( A ) RT-PCR showing normalized expression of miR-181a-5p in vector control ( RB1 -null) and RB1 -overexpressed Y79 retinoblastoma cells. ( B ) Immunoblot showing the expression of the EMT and chemo-resistant markers in vector control ( RB1 null) and RB1 -overexpressed Y79 cells. ( C ) Schematic showing the EMT program and drug resistance induction in metastatic tumors. ( D ) Phase contrast microscopy images showing the morphology of parental and resistant Y79 cells under increasing doses of topotecan treatments from week 1 to week 3. Scale bar: 400 µm. ( E ) Trypan blue cell count of parental, topotecan-resistant, and carboplatin-resistant Y79 cells for 24 h, 48 h, 72 h, and 96 h. MDR1 surface expression analysis in ( F ) parental and ( G ) topotecan-resistant Y79 cells by flow cytometry. ( H ) Bar graph showing the percentage of cells positive for MDR1 surface expression in parental, topotecan-resistant, and carboplatin-resistant Y79 cells. ( I ) Parental and resistant Y79 spheroids showing the expression of ZEB1 and cathepsin L. Scale bar: 100 µm. RT-PCR showing expression of ( J ) ZEB1, ( K ) cathepsin L, ( L ) E-cadherin, and ( M ) N-cadherin in parental, topotecan-resistant, and carboplatin-resistant Y79 cells. ( N ) Transwell invasion and migration assay to assess the migratory capacity of resistant cells compared to sensitive cells under a 10 nM topotecan treatment for 48 h. ( O ) Crystal violet OD reading at 570 nm to assess invasiveness. ( P ) Trypan blue count to assess migrated cells in the lower compartment of the transwell chamber. Two-tailed Student’s t -test (for 2 groups) and one-way ANOVA with Dunnett’s multiple comparisons tests (for >2 groups) were used for the statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. ‘ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The cell surface expression of MDR1 in parental and resistant Y79 and WERI-Rb1 cells was detected using an anti-MDR1 antibody (1:500, cat#13342, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Western Blot, Microscopy, Cell Counting, Flow Cytometry, Migration, Two Tailed Test

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Resistance depletion by miR-181a-5p conferred sensitivity to chemotherapy. ( A ) Immunoblot showing the expression of key EMT factors and drug resistance markers upon miR-181a-5p overexpression and inhibition in topotecan-resistant Y79 cells. Uncropped immunoblots are provided in . ( B ) Immunofluorescence showing the MDR1 surface expression in topotecan-resistant Y79 cells upon miR-181a-5p overexpression and inhibition. Scale bar: 50 µm. ( C ) Bar graphs showing the MDR1 fluorescent intensity in topotecan-resistant Y79 cells upon miR-181a-5p overexpression and inhibition. ( D ) Trypan blue cell count showing the proliferation of topotecan-resistant cells at 24 h, 48 h, 72 h, and 96 h upon miR-181a-5p overexpression and inhibition. ( E ) Transwell invasion and migration assay to assess the invasive and migratory capacity of topotecan-resistant cells upon miR-181a-5p overexpression and inhibition. ( F ) Crystal violet OD measurement at 570 nm to assess the invasiveness of resistant Y79 cells. ( G ) Trypan blue cell count showing the migrated cells in the lower compartment of the transwell chamber. Chemosensitivity of miR-181a-5p modulated topotecan-resistant Y79 cells upon 10 nM topotecan treatment for ( H ) 24 h, ( I ) 48 h, ( J ) 72 h, and ( K ) 96 h. The control represents untreated topotecan-resistant Y79 cells. Two-tailed Student’s t -test (for 2< group) and one-way ANOVA with Dunnett’s multiple comparisons tests (for >2 groups) were used for the statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. ‘ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The cell surface expression of MDR1 in parental and resistant Y79 and WERI-Rb1 cells was detected using an anti-MDR1 antibody (1:500, cat#13342, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing, Over Expression, Inhibition, Immunofluorescence, Cell Counting, Migration, Two Tailed Test

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Transcriptomic profiling identified differentially regulated miRNAs, EMTs, and drug resistance genes in Rb tumor subtypes. ( A ) Volcano plot showing differentially regulated miRNAs in Rb subjects ( n = 9) compared with the pediatric retinas ( n = 2) identified using a microarray. ( B ) Heatmap showing differential expression of miRNAs in 9 Rb subjects and 2 pediatric controls identified using a microarray. ( C ) Bubble scatter plot showing the top enriched KEGG pathways regulated by miRNAs in Rb tumors. ( D ) RT-PCR results showing the normalized expression of miR-181a-5p in the control retinas ( n = 4), advanced Rb ( n = 4), and non-advanced Rb ( n = 4). Volcano plot showing the differentially regulated EMT and chemotherapy resistance genes identified using a microarray in ( E ) advanced Rb tumors and ( F ) non-advanced Rb tumors. ( G ) Heatmap showing the expression of EMT and chemotherapy resistance genes in 9 Rb subjects and 2 pediatric controls. RT-PCR showing the normalized expression of ( H ) ZEB1 , ( I ) SNAI2 , ( J ) ABCB1 , and ( K ) CTSL in control pediatric retinas ( n = 4), advanced Rb tumors ( n = 4), and non-advanced Rb tumors ( n = 4). Values represent the mean ± s.d. Two-tailed Mann–Whitney was used for the statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. ’ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The separated proteins on the gel were transferred onto a PVDF membrane and were probed for specific antibodies against Rb (cat# 9309; Cell Signaling Technology, Danvers, MA, USA) phospho-Rb (cat# 8516 Cell Signaling Technology, Danvers, MA, USA), E2F1 (1:500, cat#sc193, SantaCruz Biotechnology, Dallas, TX, USA), ZEB1 (1:1000; cat#3396, Cell Signaling Technology, Danvers, MA, USA), Cathepsin L (1:1000; cat#ab6314, Abcam, Cambridge, UK), Slug (1:1000; cat#9585, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1000; cat#3195, Cell Signaling Technology, Danvers, MA, USA), N-cadherin (1:1000; cat#13116, Cell Signaling Technology, Danvers, MA, USA), MDR1 (1:1000, cat#13342, Cell Signaling Technology, Danvers, MA, USA), Total smad2/3 (1:1000, cat#ab207447, Abcam, Cambridge, UK), phospho-SMAD2 (1:1000, cat#ab53100, Abcam, Cambridge, UK), TGFBR1 (1:1000, cat#PA1731, BosterBio Pleasanton, CA, USA), TGFBR2 (1:1000, cat#ab78419, Abcam, Cambridge, UK), α-Tubulin (1:1000, cat# 3873; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000, cat#5174; Cell Signaling Technology, Danvers, MA, USA) in 5% BSA in 1X TBST overnight at 4 °C.

Techniques: Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Validation of epithelial-to-mesenchymal transition (EMT) and chemo-drug resistance genes in Rb tumors and their correlation with miR-181a-5p. Immunofluorescence results showing the expression of ( A ) ZEB1 and CTSL. The IF mean intensity of ( B ) ZEB1 and ( C ) cathepsin L staining in advanced tumors ( n = 12), non-advanced tumors ( n = 12), and control pediatric retinas ( n = 4). Immunofluorescence results showing the expression of ( D ) N-cadherin and E-cadherin, IF mean intensity of ( E ) N-cadherin and ( F ) E-cadherin in advanced tumors ( n = 12), non-advanced tumors ( n = 12), and control pediatric retina tissues ( n = 4). Scale bar: 50 µm. ( G ) Network map showing the predicted interaction of miRNA–mRNA targets using miRNet. Correlation plot showing the ( H ) negative correlation of EMT genes ( ZEB1, SNAI2, and TWIST ) with miR-181a-5p in Rb tumors and ( I ) negative correlation of drug resistance genes ( MDR1, MRP1 , and CTSL ) with miR-181a-5p in Rb tumors. Values represent the mean ± s.d. Two-tailed Mann–Whitney was used for statistical analysis. ** p < 0.01, *** p < 0.001. ’ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The separated proteins on the gel were transferred onto a PVDF membrane and were probed for specific antibodies against Rb (cat# 9309; Cell Signaling Technology, Danvers, MA, USA) phospho-Rb (cat# 8516 Cell Signaling Technology, Danvers, MA, USA), E2F1 (1:500, cat#sc193, SantaCruz Biotechnology, Dallas, TX, USA), ZEB1 (1:1000; cat#3396, Cell Signaling Technology, Danvers, MA, USA), Cathepsin L (1:1000; cat#ab6314, Abcam, Cambridge, UK), Slug (1:1000; cat#9585, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1000; cat#3195, Cell Signaling Technology, Danvers, MA, USA), N-cadherin (1:1000; cat#13116, Cell Signaling Technology, Danvers, MA, USA), MDR1 (1:1000, cat#13342, Cell Signaling Technology, Danvers, MA, USA), Total smad2/3 (1:1000, cat#ab207447, Abcam, Cambridge, UK), phospho-SMAD2 (1:1000, cat#ab53100, Abcam, Cambridge, UK), TGFBR1 (1:1000, cat#PA1731, BosterBio Pleasanton, CA, USA), TGFBR2 (1:1000, cat#ab78419, Abcam, Cambridge, UK), α-Tubulin (1:1000, cat# 3873; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000, cat#5174; Cell Signaling Technology, Danvers, MA, USA) in 5% BSA in 1X TBST overnight at 4 °C.

Techniques: Immunofluorescence, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Chemotherapy-resistant Rb cells conferred a high-EMT program and metastasis. ( A ) RT-PCR showing normalized expression of miR-181a-5p in vector control ( RB1 -null) and RB1 -overexpressed Y79 retinoblastoma cells. ( B ) Immunoblot showing the expression of the EMT and chemo-resistant markers in vector control ( RB1 null) and RB1 -overexpressed Y79 cells. ( C ) Schematic showing the EMT program and drug resistance induction in metastatic tumors. ( D ) Phase contrast microscopy images showing the morphology of parental and resistant Y79 cells under increasing doses of topotecan treatments from week 1 to week 3. Scale bar: 400 µm. ( E ) Trypan blue cell count of parental, topotecan-resistant, and carboplatin-resistant Y79 cells for 24 h, 48 h, 72 h, and 96 h. MDR1 surface expression analysis in ( F ) parental and ( G ) topotecan-resistant Y79 cells by flow cytometry. ( H ) Bar graph showing the percentage of cells positive for MDR1 surface expression in parental, topotecan-resistant, and carboplatin-resistant Y79 cells. ( I ) Parental and resistant Y79 spheroids showing the expression of ZEB1 and cathepsin L. Scale bar: 100 µm. RT-PCR showing expression of ( J ) ZEB1, ( K ) cathepsin L, ( L ) E-cadherin, and ( M ) N-cadherin in parental, topotecan-resistant, and carboplatin-resistant Y79 cells. ( N ) Transwell invasion and migration assay to assess the migratory capacity of resistant cells compared to sensitive cells under a 10 nM topotecan treatment for 48 h. ( O ) Crystal violet OD reading at 570 nm to assess invasiveness. ( P ) Trypan blue count to assess migrated cells in the lower compartment of the transwell chamber. Two-tailed Student’s t -test (for 2 groups) and one-way ANOVA with Dunnett’s multiple comparisons tests (for >2 groups) were used for the statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. ‘ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The separated proteins on the gel were transferred onto a PVDF membrane and were probed for specific antibodies against Rb (cat# 9309; Cell Signaling Technology, Danvers, MA, USA) phospho-Rb (cat# 8516 Cell Signaling Technology, Danvers, MA, USA), E2F1 (1:500, cat#sc193, SantaCruz Biotechnology, Dallas, TX, USA), ZEB1 (1:1000; cat#3396, Cell Signaling Technology, Danvers, MA, USA), Cathepsin L (1:1000; cat#ab6314, Abcam, Cambridge, UK), Slug (1:1000; cat#9585, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1000; cat#3195, Cell Signaling Technology, Danvers, MA, USA), N-cadherin (1:1000; cat#13116, Cell Signaling Technology, Danvers, MA, USA), MDR1 (1:1000, cat#13342, Cell Signaling Technology, Danvers, MA, USA), Total smad2/3 (1:1000, cat#ab207447, Abcam, Cambridge, UK), phospho-SMAD2 (1:1000, cat#ab53100, Abcam, Cambridge, UK), TGFBR1 (1:1000, cat#PA1731, BosterBio Pleasanton, CA, USA), TGFBR2 (1:1000, cat#ab78419, Abcam, Cambridge, UK), α-Tubulin (1:1000, cat# 3873; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000, cat#5174; Cell Signaling Technology, Danvers, MA, USA) in 5% BSA in 1X TBST overnight at 4 °C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Western Blot, Microscopy, Cell Counting, Flow Cytometry, Migration, Two Tailed Test

Journal: Cancers

Article Title: Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a

doi: 10.3390/cancers14205124

Figure Lengend Snippet: Resistance depletion by miR-181a-5p conferred sensitivity to chemotherapy. ( A ) Immunoblot showing the expression of key EMT factors and drug resistance markers upon miR-181a-5p overexpression and inhibition in topotecan-resistant Y79 cells. Uncropped immunoblots are provided in . ( B ) Immunofluorescence showing the MDR1 surface expression in topotecan-resistant Y79 cells upon miR-181a-5p overexpression and inhibition. Scale bar: 50 µm. ( C ) Bar graphs showing the MDR1 fluorescent intensity in topotecan-resistant Y79 cells upon miR-181a-5p overexpression and inhibition. ( D ) Trypan blue cell count showing the proliferation of topotecan-resistant cells at 24 h, 48 h, 72 h, and 96 h upon miR-181a-5p overexpression and inhibition. ( E ) Transwell invasion and migration assay to assess the invasive and migratory capacity of topotecan-resistant cells upon miR-181a-5p overexpression and inhibition. ( F ) Crystal violet OD measurement at 570 nm to assess the invasiveness of resistant Y79 cells. ( G ) Trypan blue cell count showing the migrated cells in the lower compartment of the transwell chamber. Chemosensitivity of miR-181a-5p modulated topotecan-resistant Y79 cells upon 10 nM topotecan treatment for ( H ) 24 h, ( I ) 48 h, ( J ) 72 h, and ( K ) 96 h. The control represents untreated topotecan-resistant Y79 cells. Two-tailed Student’s t -test (for 2< group) and one-way ANOVA with Dunnett’s multiple comparisons tests (for >2 groups) were used for the statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. ‘ns’ represents no statistically significant difference between the means of two variables.

Article Snippet: The separated proteins on the gel were transferred onto a PVDF membrane and were probed for specific antibodies against Rb (cat# 9309; Cell Signaling Technology, Danvers, MA, USA) phospho-Rb (cat# 8516 Cell Signaling Technology, Danvers, MA, USA), E2F1 (1:500, cat#sc193, SantaCruz Biotechnology, Dallas, TX, USA), ZEB1 (1:1000; cat#3396, Cell Signaling Technology, Danvers, MA, USA), Cathepsin L (1:1000; cat#ab6314, Abcam, Cambridge, UK), Slug (1:1000; cat#9585, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1000; cat#3195, Cell Signaling Technology, Danvers, MA, USA), N-cadherin (1:1000; cat#13116, Cell Signaling Technology, Danvers, MA, USA), MDR1 (1:1000, cat#13342, Cell Signaling Technology, Danvers, MA, USA), Total smad2/3 (1:1000, cat#ab207447, Abcam, Cambridge, UK), phospho-SMAD2 (1:1000, cat#ab53100, Abcam, Cambridge, UK), TGFBR1 (1:1000, cat#PA1731, BosterBio Pleasanton, CA, USA), TGFBR2 (1:1000, cat#ab78419, Abcam, Cambridge, UK), α-Tubulin (1:1000, cat# 3873; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000, cat#5174; Cell Signaling Technology, Danvers, MA, USA) in 5% BSA in 1X TBST overnight at 4 °C.

Techniques: Western Blot, Expressing, Over Expression, Inhibition, Immunofluorescence, Cell Counting, Migration, Two Tailed Test