Journal: International Journal of Biological Sciences

Article Title: Attenuation of STAT3 Phosphorylation Promotes Apoptosis and Chemosensitivity in Human Osteosarcoma Induced by Raddeanin A

doi: 10.7150/ijbs.30168

Figure Lengend Snippet: RA inhibits the STAT3 phosphorylation and reduces MDR1 expression in drug-resistant OS. (A) MDR1, MRP1, STAT3, and p-STAT3Tyr705 levels were determined by immunoblotting in drug-resistant cells compared with drug-sensitive cells (KHOSR and HOS, U2OSR and U2OS). (B) KHOSR and U2OSR cells were treated with vehicle or the indicated concentration of RA for 48 h. MDR1 and MRP1 expression were detected by immunoblotting. (C) MDR1 expression was determined by real-time qPCR in KHOSR and U2OSR after RA treated. (D) U2OSR and MG63 cells were transfected with pCDNA or pGMSTAT3 for 48 h and then treated with puromycin to establish a STAT3-overexpressing cell line (MG63-STAT3 and U2OSR-STAT3). MDR1 expression was detected by immunoblotting. (E) STAT3, p-STAT3Tyr705, and MDR1 levels were determined by immunoblotting in STAT3-overexpressing cell lines treated with series concentrations of RA. (F) U2OSR cells were treated with the indicated concentration of RA with or without STAT3 siRNA transfection for 48 h, and MDR1, STAT3, and p-STAT3Tyr705 expression were determined by immunoblotting. (G) MG63 cells were transfection with NC/STAT3 siRNA or pcDNA3/pGMSTAT3 for 48 h, treated with or without doxorubicin for 48h. The cell viability was detected by CCK8 assay.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-actin (Santa Cruz Biotechnology, CA, USA) and anti-PCNA, anti-caspase-3, anti-Bcl-xl, anti-Bcl-2, anti- PARP, anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-JAK2, anti-phospho-JAK2 (Tyr1007/1008), anti- Src, anti-phospho-Src (Tyr416), anti-MDR1, anti-MRP1 (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Transfection, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: Attenuation of STAT3 Phosphorylation Promotes Apoptosis and Chemosensitivity in Human Osteosarcoma Induced by Raddeanin A

doi: 10.7150/ijbs.30168

Figure Lengend Snippet: RA reverses doxorubicin resistance in human OS cells by inhibiting STAT3 phosphorylation. (A) Cells were then treated with the indicated concentration of RA for 2 hours and then incubated with calcein AM for 30 min, calcein AM efflux was evaluated by green fluorescence observed using a fluorescence microscope and quantified by SpectraMax® M5/M5e plate reader. Cells were treated with the indicated concentrations of RA for 2 hours and doxorubicin, and doxorubicin uptake was evaluated by red fluorescence observed in fluorescence images and quantified by SpectraMax® M5/M5e plate reader. The cell nucleuses were stained by DAPI, which produced blue fluorescence. Relative fluorescence activity meaned the ratio of green (or red) quantity related to blue quantity. (B) KHOSR and U2OSR cells were treated with RA in combination with the indicated concentration of doxorubicin for 48 h, and cell viability was determined by CCK8 assay. (C) U2OSR cells were treated with or without doxorubicin pretreated with or without of RA for 2 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. (D) MDR1, MRP1, STAT3 phosphorylation, total STAT3, and cleaved-PARP expression were detected by immunoblotting in U2OSR cells treated with doxorubicin in presence or absence of RA. β-actin was used as a loading control. *P<0.05, **P<0.01 compared with vehicle control.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-actin (Santa Cruz Biotechnology, CA, USA) and anti-PCNA, anti-caspase-3, anti-Bcl-xl, anti-Bcl-2, anti- PARP, anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-JAK2, anti-phospho-JAK2 (Tyr1007/1008), anti- Src, anti-phospho-Src (Tyr416), anti-MDR1, anti-MRP1 (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Concentration Assay, Incubation, Fluorescence, Microscopy, Staining, Produced, Activity Assay, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Augmented Therapeutic Potential of EC-Synthetic Retinoids in Caco-2 Cancer Cells Using an In Vitro Approach

doi: 10.3390/ijms23169442

Figure Lengend Snippet: Western blotting analysis of ABCB1 ( MDR1 ), ABCC1 ( MRP1 ) and Hsp70 in Caco-2 cells. ( A ) Representative immunoblotting images demonstrate the effect of IC 50 dose of ATRA, EC19, and EC23 on the expression levels of the indicated proteins in Caco-2 cells treated for 24 h. The expression of the investigated proteins in treated Caco-2 cells was calculated and normalized to the β-actin protein level in each treated sample, and finally plotted as ( B ) the mean protein fold change from negative control (0.1%DMSO) (dotted line) ± SEM. It can be clearly seen that EC19 and E23 caused significant downregulation of all proteins in comparison to the 0.1% DMSO control. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate a statistically significant difference. ns; non-significant.

Article Snippet: The membranes were then blocked, washed and incubated with antibodies against MDR1 (1:1000, #12683; Cell Signaling Technology, Danvers, MA, USA), MRP1 (1: 1500, #72202, Cell Signaling Technology), Hsp70 (1:1000, #MA3-009, Thermo Scientific, Waltham, MA, USA) and β-actin (1:3000, #A5060, Sigma-Aldrich, St. Louis, MO, USA) for 16–17 h at 4 °C in a humidified chamber.

Techniques: Western Blot, Expressing, Negative Control, Comparison, Control

Journal: Chemical research in toxicology

Article Title: ATF3 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an N 6 ‑Methyladenosine-Based Epitranscriptomic Mechanism

doi: 10.1021/acs.chemrestox.1c00206

Figure Lengend Snippet: ATF3 transcriptionally regulates ABCB1 in TamR MCF-7 cells. (A) Western blot images and (B) quantification results for examining the levels of ABCB1 protein in MCF-7 and TamR MCF-7 cells, TamR MCF-7 cells after knockdown of ATF3, or TamR MCF-7 cells upon the overexpression of YTHDF2 and its W432A mutant. The relative levels of ABCB1 protein were measured from band intensities using ImageJ and are depicted relative to the level detected for control cells. (C) Relative levels of ABCB1 mRNA in parental and TamR MCF-7 cells or in TamR MCF-7 cells after knockdown of ATF3. (D) TamR MCF-7 cells with a stable knockdown of ABCB1 were treated with different concentrations of 4-OHT for 72 h, and cell viability was measured using CCK8. The data were normalized against the control groups (ethanol) and represented as mean ± SD ( n = 3). (E) ChIP-qPCR data showed thw occupancy of ATF3 in the enhancer regions of ABCB1 genes in TamR MCF-7 cells. The p -values were calculated on the basis of unpaired, two-tailed Student’s t -test: *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, p < 0.001; ns, not significant (i.e., p > 0.05).

Article Snippet: Primary antibodies employed for Western blot analysis included those recognizing human ATF3 (Santa Cruz Biotechnology, sc-81189), YTHDF2 (EMD Millipore, Q2490423), ABCB1 (ABclonal, A11747), and AK4 (Santa Cruz Biotechnology, sc-271161).

Techniques: Western Blot, Knockdown, Over Expression, Mutagenesis, Control, ChIP-qPCR, Two Tailed Test

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Figure Lengend Snippet: Demographic information of the donors used to investigate regional ATP Binding Cassette B1 (ABCB1) expression in the liver.

Article Snippet: Primary antibody was rabbit polyclonal anti-ABCB1 (ab129450, Abcam, Toronto, ON, Canada) incubated for 2 h at room temperature.

Techniques: Binding Assay, Expressing

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Figure Lengend Snippet: Expression of ABCB1 within different human liver regions. ( a ) Diagram of the different regions where samples taken within the same liver; ( b ) The mRNA expression of ABCB1 was determined within different regions of 14 different livers; ( c ) Protein expression of ABCB1 within different human liver regions: (distal large lobe n = 8; distal small lobe n = 6), medial large lobe n = 7; medial small lobe n = 6, central large lobe n = 10, central small lobe n = 5, APC = adjacent to portal circulation n = 6 and ABD = adjacent to bile duct N = 11); ( d ) Correlation between mRNA expression and protein expression for ABCB1, with 95 % confidence intervals (dotted line), and data analyzed by Spearman’s correlation; ( e ) ABCB1 mRNA expression (line = means) within the livers of 10 individuals; ( f ) The ABCB1 protein expression showing intra-individual variability of protein expression (8 regions for A, D, E, J; 7 regions for B and C; 4 regions for I and 2 regions for F, G and H).

Article Snippet: Primary antibody was rabbit polyclonal anti-ABCB1 (ab129450, Abcam, Toronto, ON, Canada) incubated for 2 h at room temperature.

Techniques: Expressing

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Figure Lengend Snippet: The protein expression of ABCB1 in a cohort of 24 liver lysates. The ABCB1 protein was detected by Western blotting and normalized to the ABCB1 levels detected in a pooled S9 liver sample (Xenotech, n = 200 individuals); ( a ) correlation between age and ABCB1 protein expression with dotted lines 95% confidence intervals; ( b ) Expression of ABCB1 protein compared with BMI; ( c ) The expression of ABCB1 proteins compared to sex; ( d ) Differences between ethnicity and ABCB1 protein expression.

Article Snippet: Primary antibody was rabbit polyclonal anti-ABCB1 (ab129450, Abcam, Toronto, ON, Canada) incubated for 2 h at room temperature.

Techniques: Expressing, Western Blot

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Figure Lengend Snippet: The mRNA expression of ABCB1 in a cohort of 80 liver samples. Gene expression was measured using SYBRGreen detection and ABCB1 gene expression was normalized to 18S to give Δ C T value. ( a ) Line graph shows linear regression with 95% confidence intervals, (dotted line), of age compared to gene expression. Data analyzed by Pearson’s correlation (* p < 0.05); ( b ) Dot blot compare gene expression levels with age grouped into pediatric (≤18 years), adult (19–64 years), and geriatric (≥65 years) and data showing mean (horizontal line). The mRNA expression was also compared to ( c ) obesity, measured by BMI, ( d ) Ethnicity, and ( e ) Sex.

Article Snippet: Primary antibody was rabbit polyclonal anti-ABCB1 (ab129450, Abcam, Toronto, ON, Canada) incubated for 2 h at room temperature.

Techniques: Expressing, Dot Blot

Journal:

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Figure Lengend Snippet: Protein expression of ABCB1 in a liver cohort. The ABCB1 protein was detected by Western blotting and normalized to the ABCB1 levels detected in a pooled S9 liver sample (Xenotech, n = 200 individuals). ( a ) Example of Western blot of 6 individuals with 20 µg liver S9 loaded compared with pooled S9 sample, ABCB1 = recombinant ABCB1 expressed in baculosome (5 µg), and blank = baculosome with no expression (5 µg); ( b ) Relationship between age and ABCB1 protein expression with a one-phase association, with 95% confidence intervals (dotted line); ( c ) Protein expression compared to individuals grouped by age and analyzed by ANOVA ( p < 0.0001) and ABCB1 expression in pediatrics was significantly lower compared to adults and geriatrics (Tukey’s multiple comparison test, *** p < 0.0001, * p < 0.05); ( d ) Expression of ABCB1 protein in obese individuals, measured by BMI; ( e ) Differences between ethnicity and ABCB1 protein expression; ( f ) The expression of ABCB1 proteins compared to sex.

Article Snippet: Primary antibody was rabbit polyclonal anti-ABCB1 (ab129450, Abcam, Toronto, ON, Canada) incubated for 2 h at room temperature.

Techniques: Expressing, Western Blot, Recombinant