P Glycoprotein 1（MDR1） Antibody - #AF5185

Figure 4. Cytotoxicity of different drug formulations. (a) MTT assay of HepG2 cells and the cocultured model. (b) Cell viability of HepG2 cells. (c) Cell viability of the cocultured system. Scale bars represent 100 μm. (d) Live/dead staining of HepG2 cells, where green- and red-dyed cells represent live and dead HepG2 cells, respectively. (e) Live/dead staining of the cocultured model, where the yellow-, red-, and green-colored cells denote the dead LX-2, dead HepG2, and live LX-2 cells, respectively. Scale bars represent 100 μm. (f) Western blotting assay for the expression of P-gp. (g) Quantitative analysis of P-gp. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2| Autophagy is increased and apoptosis is reduced in the EOC re-ascites group. a IHC analysis of LC-3, Belin-1, CASP-9, and c-CASP-3 expression in EOC ascites cells (scale bar =30µm). b, c Western blot analysis of autophagy/apoptosis-related proteins expressed in the chemosensitive group and re-ascites group (***p<0.001, **p<0.01, *p<0.05). d Immunofluorescence analysis of the chemosensitive group and re-ascites group with LC3 (green) and LAMP2 (red) staining. Nuclei were counterstained with DAPI (blue). e qRT-PCR analysis of the average relative mRNA expression levels of autophagy/apoptosis-related genes in the chemosensitive group and re-ascites group (**p<0.01, *p<0.05)

FIGURE 3  miR-506 down-regulated MDR1/P-gp expression in HCT116-OxR. A, The mRNA level of MDR1 was decreased after transfection with the miR-506 mimic of the relative chemoresistant genes as demonstrated by qRT-PCR. B, The protein level of MDR1 was decreased after transfection with the miR-506 mimic of the relative chemoresistant proteins as demonstrated by Western blot. C, Expression of P-gp detected by immunofluorescence staining.HCT116-OxR-miR-506 cells showed low levels of fluorescent staining of P-gp, whereas maximal staining of P-gp was observed in HCT116-OxR cells, readily distinguished from background. Zoom: 200×. *P<.05)

Figure 6 Western blotting against α-SMA, Bcl-2, and P-gp in total soluble protein fractions from (1) control 4T1 cells in monoculture, (2) control co-cultures of 4T1 cells and NIH 3T3 fibroblasts, or co-cultures treated with (3) free CEL, (4) free CEL+BA, (5) CL, (6) CL@BM or (7) F/CL@BM. (A) Western blotting assay for the expression of protein. (B) Quantitative analysis. ***p < 0.001, ****p < 0.0001.

Figure 2 DHW-221 accelerates intracellular Rho-123 accumulations and inhibits P-gp expression by binding to P-gp. (A) Comparison of P-gp in A549 and A549/Taxol cells. Statistical comparisons were performed with unpaired Student’s t-test (n = 3). **p < 0.01 versus A549. (B, C) After treatment of DHW-221 or verapamil for 48 h, Rho-123 (15 µM) was added into the mediums to co-incubate with DHW-221 or verapamil for 3 h in dark. The intracellular accumulation of Rho-123 was evaluated by flow cytometry and confocal microscope, respectively. Verapamil (VRP, 10 μM) was used as a positive control. Green fluorescence indicated Rho-123, and blue fluorescence indicated DAPI. Scale bar = 100 μm. Nuclear mean fluorescence intensity of Rho 123 was measured by ImageJ and analyzed using FlowJo 10 software. Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control. (D) Effect of DHW-221 or verapamil to reverse Taxol resistance for 48 h in A549/Taxol cells. (E) Western blot analysis of DHW-221 inhibited P-gp expression in A549/Taxol cells. (F) Ribbon diagram of human ABCB1 (PDB code: 7A6E, purple spiral) bound to drug (membrane region) and predicted binding modes for DHW-221 (blue stick) and tariquidar (orange stick, a known third-generation P-gp inhibitor) with ABCB1. Hydrogen bonds were shown as green dashed lines. Key residues for DHW-221 and tariquidar interaction were highlighted. (G) Effect of DHW-221 on the thermal stability of ABCB1 was quantitatively detected by a cellular thermal shift assay (CETSA). Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons (n = 3). Data are presented as mean ± SD. **p < 0.01, ****p < 0.0001versus control.