Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Doxorubicin and TQR combination studies in the MDA-MB-468 MDR1 cell line. Timecourses of the mean intracellular concentration of doxorubicin with corresponding standard deviations are shown for each treatment condition in (A) . Doxorubicin accumulation increases along with TQR concentrations. Equations (1–3) were fit to the data, and the best-fit models are overlaid on the data (smooth lines) in a. Model parameter fits corresponding to the best-fit models are shown in (B–D) . Similar k EF and k FB vales are observed across all TQR concentrations. There is a trend of decreasing k FE values with increasing TQR concentrations (C) , consistent with MDR1 inhibition by TQR. Cell counts of MDA-MB-468 MDR1 following combination treatment with TQR and doxorubicin are show in panels (E-G) . In each plot, a fixed concentration of doxorubicin is applied with variable TQR concentrations. These counts are fit with Equations (4–6) as described in section Model Fits, and the best-fit model is overlaid on the cell counts [smooth lines in panels (E–G) ]. Error bars represent the 95% CI from six experimental replicates for each treatment condition. Model parameters with corresponding 95% CI are shown in (H–J) as a function of TQR concentration. For each doxorubicin concentration, the death rate ( k d,a and k d,b ) increased with TQR concentration (H,I) .

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Treatment response in MDA-MB-468 MDR1 (left column) and SUM-149PT (right column) cell lines under doxorubicin monotherapy. The top row [panels (A,E) ] shows cell counts over time from treatment response studies for each cell line. For these studies, cells were treated with a fixed concentration of doxorubicin for 24 h. These counts are fit to Equations (4–6) as described in section Model Fits, and the best-fit model is overlaid on the cell counts [smooth lines in (A,E) ]. Error bars represent the 95% CI from six experimental replicates for each treatment condition. Model parameters with corresponding 95% CI are shown in the bottom three rows as a function of doxorubicin concentration. Panels (B–D) show fits from the MDA-MB-468 MDR1 experiments, and panels (F–H) show fits from the SUM-149PT experiments. For each doxorubicin concentration for each cell line, the death rate ( k d,a and k d,b ) increased with increasing doxorubicin concentrations.

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Leveraging equivalent dose to estimate the effect of TQR in the MDA-MB-468 MDR1 cell line. The equivalent dose for each doxorubicin monotherapy condition was first calculated with the PK model parameters measured in the doxorubicin uptake studies. The equivalent dose statistic was then estimated for each co-treatment condition by matching treatment response parameters from co-treatment conditions to those from doxorubicin monotherapy conditions. Parameter values from all doxorubicin monotherapy and co-treatment conditions are plotted as a function of equivalent dose (A-C) . A subset of responses from doxorubicin monotherapy and co-treatment conditions are color-coded to their estimated equivalent dose (D–F) . Similar dynamics are observed with similarly-colored data, demonstrating the efficacy of the parameter matching in comparing treatment response timecourses. As TQR impairs the function of the MDR1 pump, we hypothesized the effect of TQR is limited to the k FE parameter (G) . With estimates of equivalent dose for all treatment conditions, the k FE value for each TQR concentration was estimated with the optimization routine summarized by Equation (7) (H) . These values, calculated with treatment response data, agree well with direct measurements of k FE reported in . We note the large confidence intervals are a result of the optimization approach, in which the value (1/ k FE ) was optimized.

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Comparison of MDA-MB-468 H2B and MDA-MB-468 MDR1 cell lines using equivalent dose. The intracellular doxorubicin concentration with 95% CI for each cell line is shown in (A) . The MDA-MB-468 H2B line demonstrates increased intracellular accumulation of doxorubicin relative to the MDA-MB-468 MDR1 line. Equations (1–3) are fit to the doxorubicin uptake data, and the best-fit models are overlaid on the data in a (smooth line). The corresponding parameters with 95% CI are shown in (B–D) . The MDA-MB-468 H2B data are shown in red, and the MDA-MB-468 MDR1 data are shown in blue. Notably, the efflux of drug from the MDA-MB-468 MDR1 ( k FE ) line is significantly greater than the corresponding rate in the MDA-MB-468 H2B line ( p < 0.05). Treatment response is traditionally summarized by cell survival and plotted against applied drug concentration. The cell count relative to control for each cell line is shown as a function of extracellular doxorubicin concentration and equivalent dose in (E,F) , respectively. While a significant difference is observed when comparing these cell lines via EC 50 calculated with the extracellular doxorubicin concentration, no significant difference is observed when comparing the EC 50 statistic derived from the equivalent dose. The equivalent dose can account for the differing pharmacokinetic properties to reveal similar doxorubicin pharmacodynamics in these cell lines.

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay, Cell Counting, Derivative Assay

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: CircARVCF knockdown suppressed DDP resistance in DDP-resistant GC cells. (A) The expression of circARVCF in MKN-45/DDP and AGS/DDP cells transfected with si-circARVCF#1, si-circARVCF#2, si-circARVCF#3, or si-NC was detected by qRT-PCR. (B–I) MKN-45/DDP and AGS/DDP cells were transfected with si-NC or si-circARVCF#1. (B,C) IC50 of cisplatin in MKN-45/DDP and AGS/DDP cells was examined with CCK-8 assay. (D) The colony formation of MKN-45/DDP and AGS/DDP cells was evaluated by colony formation assay. (E,F) The migration and invasion of MKN-45/DDP and AGS/DDP cells were tested by transwell assay. (G) The apoptosis of MKN-45/DDP and AGS/DDP cells was analyzed by flow cytometry analysis. (H,I) The protein levels of MRP1, MDR1, Bcl-2 and Bax in MKN-45/DDP and AGS/DDP cells were measured via western blot assay. * p < 0.05.

Article Snippet: The antibodies included multidrug resistance protein 1 (MRP1; bs-24241R; 1:2000; Bioss), p-glycoprotein (MDR1; bs-0563R; 1:2000; Bioss), B-cell lymphoma-2 (Bcl-2; bs-20351R; 1:2000; Bioss), BCL2-Associated X (Bax; bs-28034R; 1:2000; Bioss), FGFR1 (bs-0230R; 1:2000; Bioss) and β-actin (bs-0061R; 1:10,000; Bioss).

Techniques: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Migration, Transwell Assay, Flow Cytometry, Western Blot

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: Inhibition of miR-1205 reversed the effects of circARVCF knockdown on DDP resistance, cell colony formation, migration, invasion, and apoptosis in DDP-resistant GC cells. MKN-45/DDP and AGS/DDP cells were transfected with si-NC, si-circARVCF#1, si-circARVCF#1+in-miR-NC, or si-circARVCF#1+in-miR-1205. (A,B) IC50 of cisplatin was estimated by CCK-8 assay. (C) The colony formation of MKN-45/DDP and AGS/DDP cells was examined with colony formation assay. (D,E) The migration and invasion of MKN-45/DDP and AGS/DDP cells were assessed by transwell assay. (F) The apoptosis of MKN-45/DDP and AGS/DDP cells was analyzed by flow cytometry analysis. (G,H) The protein levels of MRP1, MDR1, Bcl-2 and Bax in MKN-45/DDP and AGS/DDP cells were measured via western blot assay. * p < 0.05.

Article Snippet: The antibodies included multidrug resistance protein 1 (MRP1; bs-24241R; 1:2000; Bioss), p-glycoprotein (MDR1; bs-0563R; 1:2000; Bioss), B-cell lymphoma-2 (Bcl-2; bs-20351R; 1:2000; Bioss), BCL2-Associated X (Bax; bs-28034R; 1:2000; Bioss), FGFR1 (bs-0230R; 1:2000; Bioss) and β-actin (bs-0061R; 1:10,000; Bioss).

Techniques: Inhibition, Migration, Transfection, CCK-8 Assay, Colony Assay, Transwell Assay, Flow Cytometry, Western Blot

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: MiR-1205 regulated the DDP resistance and malignant behaviors of DDP-resistant GC cells by targeting FGFR1. MKN-45/DDP and AGS/DDP cells were transfected with miR-NC, miR-1205, miR-1205 + pcDNA or miR-1205 + FGFR1. (A,B) DDP resistance was analyzed by CCK-8 assay. (C–F) The colony formation, migration, invasion and apoptosis of MKN-45/DDP and AGS/DDP cells were evaluated by colony formation assay, transwell assay and flow cytometry analysis, respectively. (G,H) The protein levels of MRP1, MDR1, Bcl-2 and Bax in MKN-45/DDP and AGS/DDP cells were measured via western blot assay. * p < 0.05.

Article Snippet: The antibodies included multidrug resistance protein 1 (MRP1; bs-24241R; 1:2000; Bioss), p-glycoprotein (MDR1; bs-0563R; 1:2000; Bioss), B-cell lymphoma-2 (Bcl-2; bs-20351R; 1:2000; Bioss), BCL2-Associated X (Bax; bs-28034R; 1:2000; Bioss), FGFR1 (bs-0230R; 1:2000; Bioss) and β-actin (bs-0061R; 1:10,000; Bioss).

Techniques: Transfection, CCK-8 Assay, Migration, Colony Assay, Transwell Assay, Flow Cytometry, Western Blot

Journal: Bioorganic & medicinal chemistry letters

Article Title: Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria

doi: 10.1016/j.bmcl.2018.04.001

Figure Lengend Snippet: Compound 1 eradicates MRSA (ATCC BAA 44) in the presence of light. A: Graph, showing a dose-dependent effect of 1 with and without a 2-minute irradiation with white light. B. Drip-streak plates utilized in the experiment illustrating the CFU counts. The data are derived from a single experiment performed in 3 replicates and serve as an example of 3 independent experiments producing similar results. The stars represent no observable colonies.

Article Snippet: The panel included: another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive Staphylococcus aureus ; vancomycin-resistant Enterococcus faecalis , known as VRE and representing another bacterial genus on the WHO list as discussed above; 26 Streptococcus pyogenes , a causative agent of important human diseases, ranging from superficial skin infections to life-threatening systemic infections; 27 Streptococcus mutans , a species commonly found in human oral cavity and the main contributor to tooth decay.

Techniques: Irradiation, Derivative Assay

Journal: Bioorganic & medicinal chemistry letters

Article Title: Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria

doi: 10.1016/j.bmcl.2018.04.001

Figure Lengend Snippet: Bacterial patterning with light. Left: MRSA (ATCC BAA-44) cells pre-treated with 1 (1 μM), spread over an agar plate and irradiated with light through a mask for 2 minutes, and then incubated at 37 °C. NMT: New Mexico Tech. Right: Schematic of the bacterial patterning experiment.

Article Snippet: The panel included: another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive Staphylococcus aureus ; vancomycin-resistant Enterococcus faecalis , known as VRE and representing another bacterial genus on the WHO list as discussed above; 26 Streptococcus pyogenes , a causative agent of important human diseases, ranging from superficial skin infections to life-threatening systemic infections; 27 Streptococcus mutans , a species commonly found in human oral cavity and the main contributor to tooth decay.

Techniques: Irradiation, Incubation

Journal: Bioorganic & medicinal chemistry letters

Article Title: Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria

doi: 10.1016/j.bmcl.2018.04.001

Figure Lengend Snippet: Compound 1 is effective against a variety of drug resistant Gram-positive bacterial strains in the presence of light. A. MRSA (ATCC BAA1717). B. S. aureus (ATCC 29213). C. E. faecalis (ATCC 51299). D. S. pyogenes (ATCC 8133). E. S. mutans (Ward’s 85W 2357). The data are derived from a single experiment performed in 3 replicates and serve as an example of 3 independent experiments producing similar results. The stars represent no observable colonies.

Article Snippet: The panel included: another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive Staphylococcus aureus ; vancomycin-resistant Enterococcus faecalis , known as VRE and representing another bacterial genus on the WHO list as discussed above; 26 Streptococcus pyogenes , a causative agent of important human diseases, ranging from superficial skin infections to life-threatening systemic infections; 27 Streptococcus mutans , a species commonly found in human oral cavity and the main contributor to tooth decay.

Techniques: Derivative Assay

Journal: Bioorganic & medicinal chemistry letters

Article Title: Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria

doi: 10.1016/j.bmcl.2018.04.001

Figure Lengend Snippet: Eradication of MRSA (ATCC BAA44) by analogues of 1. The data are derived from a single experiment performed in 3 replicates and serve as an example of 3 independent experiments producing similar results. The stars represent no observable colonies.

Article Snippet: The panel included: another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive Staphylococcus aureus ; vancomycin-resistant Enterococcus faecalis , known as VRE and representing another bacterial genus on the WHO list as discussed above; 26 Streptococcus pyogenes , a causative agent of important human diseases, ranging from superficial skin infections to life-threatening systemic infections; 27 Streptococcus mutans , a species commonly found in human oral cavity and the main contributor to tooth decay.

Techniques: Analogues, Derivative Assay

Journal: Bioorganic & medicinal chemistry letters

Article Title: Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria

doi: 10.1016/j.bmcl.2018.04.001

Figure Lengend Snippet: SEM images of MRSA (ATCC BAA44) cells treated with 1.

Article Snippet: The panel included: another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive Staphylococcus aureus ; vancomycin-resistant Enterococcus faecalis , known as VRE and representing another bacterial genus on the WHO list as discussed above; 26 Streptococcus pyogenes , a causative agent of important human diseases, ranging from superficial skin infections to life-threatening systemic infections; 27 Streptococcus mutans , a species commonly found in human oral cavity and the main contributor to tooth decay.

Techniques:

Journal: Bioorganic & medicinal chemistry letters

Article Title: Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria

doi: 10.1016/j.bmcl.2018.04.001

Figure Lengend Snippet: Permeability of MRSA (ATCC BAA44) cell membrane probed by PI. Green fluorescence is due to 1. Red fluorescence is indicative of the disruption of cell membrane.

Article Snippet: The panel included: another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive Staphylococcus aureus ; vancomycin-resistant Enterococcus faecalis , known as VRE and representing another bacterial genus on the WHO list as discussed above; 26 Streptococcus pyogenes , a causative agent of important human diseases, ranging from superficial skin infections to life-threatening systemic infections; 27 Streptococcus mutans , a species commonly found in human oral cavity and the main contributor to tooth decay.

Techniques: Permeability, Membrane, Fluorescence, Disruption

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Changes in ATP Binding Cassette Subfamily B Member 1 (Abcb1) Expression in Gastrocnemius Muscle and Correlations with DCA and LCA Concentrations in the Tissue Microenvironment of the Control Group, Sarcopenia Group A, and Sarcopenia Group B. Abcb1 mRNA levels in mouse gastrocnemius muscle were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) ( a ). Representative Western blot images of P-gp and GAPDH proteins. For each target, lanes 1–8: Control Group; lanes 9–16: Sarcopenia Group A; and lanes 17–24: Sarcopenia Group B ( b ). Quantitative analysis of P-gp protein expression levels normalized to GAPDH (n = 8 per group) ( c ). Spearman’s bivariate correlation analysis was performed to assess correlations between DCA/LCA concentrations in the skeletal muscle microenvironment and P-gp expression ( d , e ). ** p < 0.01; **** p < 0.0001, p > 0.05 was considered as not significant (ns).

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: Binding Assay, Expressing, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Increased Intracellular Bile Acid Concentrations and Altered Expression of Endothelial Function Regulatory Genes in Skeletal muscle microvascular endothelial cells (MMECs) of the Valspodar Group and Si-ABCB1 Group. Intracellular DCA ( a , c ) and LCA ( b , d ) concentrations in MMECs were measured by HPLC-MS/MS. Expression of interleukin 6 (Il6) ( e , g ) and vascular endothelial growth factor A (Vegfa) ( f , h ) in MMECs was detected by qRT-PCR. VEGFA protein expression levels were analyzed by Western blot with quantification ( i , j ). * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: Expressing, Tandem Mass Spectroscopy, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Effects of conditioned medium (CM) from Different MMEC Groups on C2C12 Viability and Proliferation. C2C12 counts in the Valspodar Group, the DMSO Group, the Si-ABCB1 Group, and the Si-CON Group ( a ). C2C12 cell viability assessed by CCK-8 assay across groups ( b ). Representative EdU and DAPI staining images with statistical analysis of proliferating (EdU + ) C2C12 cell percentages ( c ). ** p < 0.01; *** p < 0.001.

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: CCK-8 Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Effects of CM from Different MMEC Groups on Inflammatory Factors and Differentiation-Related Functions in C2C12-Derived Myotubes. Expression of inflammatory factors Tumor necrosis factor-alpha (Tnf-α) ( a , k ), Interleukin 6 (Il6) ( b , l ), Interleukin 10 (Il10) ( c , m ), Interleukin 1β (Il1β) ( d , n ), and C-X-C motif chemokine ligand 1 (Cxcl1) ( e , o ) in myotubes treated with CM from the Valspodar Group, the DMSO Group, the Si-ABCB1 Group, and the Si-CON Group of MMECs was detected by RT-qPCR. Expression of myogenic key genes Myogenic factor 5 (Myf5) ( f , p ), Muscle regulatory factor 4 (Mrf4) ( g , q ), Myogenin ( h , r ), F-box protein 32 (Fbxo32) ( i , s ), and Tripartite motif containing 63 (Trim63) ( j , t ) was also analyzed. Protein expression levels of phospho-p38 (p-p38), total p38 mitogen-activated protein kinase (p38 MAPK), MYOGENIN, and myogenic differentiation 1 (MYOD1) were assessed by Western blot with quantification ( u ). * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001, p > 0.05 was considered as not significant (ns).

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Cell Characterization, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Abcb1 was targeted by miR-135a-5p in MMECs. Candidate human and mouse miRNAs predicted to target ABCB1/ Abcb1 on Tagetscan database ( a ). Expression of screened miRNAs in muscle tissues from the Control Group and Sarcopenia Group ( b ). RNA pull-down assays were conducted to detect the binding between Abcb1 and miR-582-5p ( c ) or miR-135a-5p ( d ) in MMECs. qRT-PCR was used to detect the overexpression efficiency of miR-135a-5p in MMECs ( e ). Luciferase reporter assays were performed to determine the interaction between miR-135a-5p and Abcb1 in MMECs ( f ). qRT-PCR was used to detect the expression of Abcb1 mRNA in MMECs transfected with NC mimics or miR-135a-5p mimics ( g ). ** p < 0.01; *** p < 0.001, p > 0.05 was considered as not significant (ns).

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: Expressing, Control, Binding Assay, Quantitative RT-PCR, Over Expression, Luciferase, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Effects of miR-135a-5p/ Abcb1 Axis on Bile Acid Efflux of MMECs. qRT-PCR analysis of miR-135a-5p ( a , b ) Abcb1 mRNA expression in MMECs in each group. Western blot analysis and quantification of P-gp protein expression in MMECs ( c ). Intracellular DCA ( d ) and LCA ( e ) concentrations in MMECs were measured by HPLC-MS/MS. Expression of Tnf-α ( f ), endothelial nitric oxide synthase (Enos) ( g ), and superoxide dismutase 2 (Sod2) ( h ) in MMECs was detected by qRT-PCR. VEGFA protein expression levels were analyzed by Western blot and quantified ( i ). ** p < 0.01; *** p < 0.001, p > 0.05 was considered as not significant (ns).

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Tandem Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Bile Acid Metabolism Affects Muscle Regeneration in Aging Skeletal Muscle in a Manner Associated with Regulation of ABCB1 Expression

doi: 10.3390/ijms27062649

Figure Lengend Snippet: Effects of CM from miR-135a-5p and Abcb1 -overexpressed MMECs on C2C12 cells. C2C12 cell viability was assessed by CCK-8 assays across groups ( a ). Expression of inflammatory factors Tnf-α ( b ) Il6 ( c ), Il10 ( d ), and Cxcl1 ( e ) in myotubes treated with CM from each group was detected using qRT-PCR. Expression of myogenic key genes Myf5 ( f ), Mrf4 ( g ), Myogenin ( h ), Fbxo32 ( i ), and Trim63 ( j ) was also analyzed by qRT-PCR. Representative images of immunofluorescence staining of MyHC and DAPI staining in C2C12-derived myotubes treated with CM from transfected MMECs ( k ). Scale bar = 100 μm; red: MyHC, blue: nuclei. Measured and statistically analyzed: MyHC-positive area proportion ( l ), myotube diameter ( m ), average nuclei number per myotube ( n ), and myotube fusion index ( o ). Protein expression levels of phospho-p38 (p-p38), total p38 MAPK, MYOGENIN, and MYOD1 were assessed by Western blot ( p ) with quantification ( q – s ). *** p < 0.001, ** p > 0.01, p > 0.05 was considered as not significant (ns).

Article Snippet: The membranes were blocked in 5% skim milk and incubated overnight at 4 °C with primary antibodies against ABCB1 (Proteintech Group, Inc., Rosemont, IL, USA, 22336-1-AP, 1:1000), VEGFA (Abcam, Cambridge, UK, ab46154, 1:1000), MYOGENIN (Proteintech Group, Inc., Rosemont, IL, USA, 26762-1-AP, 1:10,000), MYOD1 (Proteintech, 18943-1-AP, 1:5000), p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #8690, 1:1000), phospho-p38 MAPK (Cell Signaling Technology, Danvers, MA, USA, #4511, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Abcam, Cambridge, UK, ab8245, 1:5000).

Techniques: CCK-8 Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Derivative Assay, Transfection, Western Blot