Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: Primer sequences of OCT2, OAT1, OAT3, P-gp, MATE1, MATE2K, MRP2 genes.

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: Values of the binding energies of berberine and berberrubine to seven transporter proteins in the kidney (Kcal/mol).

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: Molecular docking illustrations of BBR and BBRR with seven transport proteins respectively. (A) BBR docked with OAT1. (B) BBR docked with OAT3. (C) BBR docked with OCT2. (D) BBR docked with MATE1. (E) BBR docked with MATE2K. (F) BBR docked with P‐gp. (G) BBR docked with MRP2. (H) BBRR docked with OAT1. (I) BBRR docked with OAT3. (J) BBRR docked with OCT2. (K) BBRR docked with MATE1. (L) BBRR docked with MATE2K. (M) BBRR docked with P‐gp. (N) BBRR docked with MRP2.

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: Trends of metformin, methotrexate, estrone-3-sulfate, topotecan, rhodamine 123, and vinblastine concentration under different dosing time conditions in cells lysates or extracellular medium from HEK-293 cells. Blank control (BC), raw Phellodendri Chinensis cortex (RPC), Phellodendri Chinensis Cortex with salt-water processing (SPC), berberine (BBR), berberrubine (BBRR). (A) The trend of MET concentration changes mediated by OCT2. (B) The trend of MTX concentration changes mediated by OAT1. (C) The trend of E3S concentration changes mediated by OAT3. (D) The trend of Rho123 concentration changes mediated by P-gp. (E) The trend of TOP concentration changes mediated by MATE1 and MATE2K. (F) The trend of VBL concentration changes mediated by MRP2 (n = 3).

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques: Concentration Assay, Control

Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: The effect of various solutions on each substrate accumulation transported by renal transport proteins. Blank control (BC), raw Phellodendri Chinensis cortex (RPC), Phellodendri Chinensis Cortex with salt-water processing (SPC), berberine (BBR), berberrubine (BBRR) (A) The effect of various solutions on the accumulation of MET transported by OCT2. (B) The effect of various solutions on the accumulation of MTX transported by OAT1. (C) The effect of various solutions on the accumulation of E3S transported by OAT3. (D) The effect of various solutions on the accumulation of Rho123 transported by P-gp. (E) The effect of various solutions on the accumulation of TOP transported by MATE1 and MATE2K. (F) The effect of various solutions on the accumulation of VBL transported by MRP2. Compared with the RPC group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; compared with the SPC group, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 (n = 3).

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: The effect of various solutions on the protein expression of MATE1, MATE2K, P-gp, MRP2. Blank control (BC), raw Phellodendri Chinensis cortex (RPC), Phellodendri Chinensis Cortex with salt-water processing (SPC), berberine (BBR), berberrubine (BBRR). (A) MATE1, MATE2K, P-gp, MRP2 protein expression bands in HEK-293 cells. (B) The effect of various solutions on MATE1 protein expression level. (C) The effect of various solutions on MATE2K protein expression level. (D) The effect of various solutions on P-gp protein expression level. (E) The effect of various solutions on MRP2 protein expression level. Compared with the BC group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; compared with the RPC group, □ p < 0.05, □□ p < 0.01, □□□ p < 0.001, □□□□ p < 0.0001; compared with the BBR group, △ p < 0.05, △△ p < 0.01, △△△ p < 0.001, △△△△ p < 0.0001.

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: A strategy for evaluating the impact of processing of Chinese meteria medica on meridian tropism: the influence of salt-water processing of phellodendri chinensis cortex on renal transport proteins

doi: 10.3389/fphar.2025.1558298

Figure Lengend Snippet: The effect of various solutions to MATE1, MATE2K, P-gp, MRP2 mRNA expression level. Blank control (BC), raw Phellodendri Chinensis cortex (RPC), Phellodendri Chinensis Cortex with salt-water processing (SPC), berberine (BBR), berberrubine (BBRR). (A) The effect of various solutions on MATE1 mRNA expression level. (B) The effect of various solutions on MATE2K mRNA expression level. (C) The effect of various solutions on P-gp mRNA expression level. (D) The effect of various solutions on MRP2 mRNA expression level Compared with the BC group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; compared with the RPC group, □ p < 0.05, □□ p < 0.01, □□□ p < 0.001, □□□□ p < 0.0001; compared with the BBR group, △ p < 0.05, △△ p < 0.01, △△△ p < 0.001, △△△△ p < 0.0001.

Article Snippet: DMEM high glucose medium (Cat. No.: 8123207), PBS buffer salt solution (Cat. No.: 20210927) were purchased from Gibco (Waltham, Massachusetts, United States), fetal bovine serum (Cat. No.: ST200913) was purchased from PAN (Aden Bach, Free State of Bavaria, Germany), penicillin-streptomycin-Amphotericin B Mixed Triple Antibody Solution (Cat. No.: 20220519JH), 20×TBST buffer (Cat. No.: T1082), Whole Protein Lysis Kit (Cat. No.: BC3710), TRIzol Lysis Solution (Cat. No.: 15596018CN), ECL PLUS Ultra-Sensitive Luminescent Solution (Cat. No.: PE0010), dimethyl sulfoxide (Cat. No.: 710N0310), metformin (Cat. No.: D9351, purity≥98%), methotrexate (Cat. No.: M8971, purity≥98%), rhodamine123 (Cat. No.: R8030, purity≥98%), topotecan (Cat. No.: IT1030, purity≥98%) were purchased from Solarbio Science and Technology Co. Ltd (Beijing, China), SDS-PAGE Protein Sampling Buffer 5× (Cat. No.: P0015L) was purchased from Beyotime Bio-Tech Ltd (Shanghai, China), OCT2 rabbit anti-polyclonal antibody (Cat. No.: 10867-2-AP), OAT1 rabbit anti-polyclonal antibody (Cat. No.: 26574-1-AP), OAT3 rabbit anti-polyclonal antibody (Cat. No.: 16844-1-AP), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20,898), P-gp rabbit anti-polyclonal antibody (Cat. No.: 22336-1-AP), MATE1 rabbit anti-polyclonal antibody (Cat. No.: 20898-1-AP), MATE2K rabbit anti-polyclonal antibody (Cat. No.: 26873-1-AP), MRP2 rabbit anti-polyclonal antibody (Cat. No.: 29261-1-AP), Goat Anti-Rabbit IgG II Antibody (Cat. No.: 20001097) were purchased from the Proteintech (Wuhan, China).

Techniques: Expressing, Control

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 1. An overall survival area plot showing different estimates of the causal effect of the ZWILCH expression on survival using different Cox proportional hazards regression models (A). The contour plot illustrates the probability of patient survival over time according to ZWILCH gene expression level in ACC patients from the TCGA database (B).

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Expressing, Gene Expression

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 5. General transcriptome profile in groups of 10 patients with the highest expression of the ZWILCH gene (up ZWILCH) compared to 10 patients with the lowest expression of the ZWILCH gene (down ZWILCH) (data from GSE10927). Each dot on the graph represents the mean value of the gene expression level of ten patients belonging to a particular group. The cut-off criteria for identifying differentially expressed genes were established as |fold change| = 1.5 and p-value = 0.05, and are represented by orange dotted lines. Genes above the cut-off lines were categorized as up−regulated (green dots) or down−regulated (red dots). The total number of up−and down−regulated genes can be found in the top right and top left corners, respectively. The plots also indicate the symbols for the seven most differentially expressed genes.

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Expressing, Gene Expression

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 8. Gene set enrichment analysis (GSEA) from the comparison of patients with low and high ZWILCH expression, based on GSE10927. Clustering of enriched gene sets into common functional groups, each cluster is marked with a different color (A). The barplot with ten of the most activated and inhibited gene terms according to the normalized enrichment score values (B). Detailed enrichment plots for the five most inhibited and the five most activated gene sets, showing the profile of the running NES score and the positions of the genes on the rank−ordered list (C,D).

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Comparison, Expressing, Functional Assay

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 10. The expression of ZWILCH protein in human adrenal gland disease spectrum (adrenal cancer progression) tissue microarray (TMA) slide. The general profile of immunohistochemical staining presents localization of ZWILCH protein (A). TMA map shows the relevant types of adrenal cancer progression marked in an appropriate color (B). Densitometric analysis of ZWILCH gene expression in the tissue array side of the adrenal gland disease spectrum. The boxplot displays each group with its median and interquartile range (IQR) (C). Individual patient densitometric data were added to the corresponding boxplots and represented as dots. The Kruskal–Wallis (KW) test was used to compare groups, followed by the Dunn post hoc test. Differences between groups were marked with asterisks: ** for p < 0.01. Letter annotation was used to indicate statistically significant differences (p < 0.05) between compared groups. Paired comparisons with post hoc Dunn p-values are also shown in the table.

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Expressing, Microarray, Immunohistochemical staining, Staining, Gene Expression

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 11. Representative immunostaining of adrenal gland disease spectrum (adrenal cancer pro- gression) TMA slide. Brown staining (B,C,E,F,H,I,K,L,N,O) indicates ZWILCH protein (red arrows), located typically cellular with hematoxylin counterstain (nucleus). The negative control of adrenal gland tissue (A,D,G,J,L,M). Original magnification was 100× (A,B,D,E,G,H,M,N), 200× (J,L,K), and 400× (C,F,I,L,O).

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Immunostaining, Staining, Negative Control

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 1. RASEF expression in tumor tissues and cell lines. A, expression of RASEF in 12 clinical lung cancers (T; 4 clinical lung ADC, 4 clinical lung SCC, and 4 clinical SCLC) and corresponding normal lung tissues (N) detected by semiquantitative RT-PCR analysis. B, expression of RASEF in 22 lung cancer cell lines and a bronchial epithelial cell line BEAS-2B detected by semiquantitative RT-PCR analysis. ASC indicates lung adenosquamous cell carcinoma; LCC, large cell carcinoma. C, Western blot analysis of RASEF protein using anti-RASEF antibody. IB, immunoblotting. D, expression and subcellular localization of endogenous RASEF protein in RASEF-positive and RASEF-negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in A549 and NCI-H2170 cells, whereas no staining was observed in DMS114 and bronchial epithelia–derived BEAS-2B cell lines.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 4. Enhanced phosphorylation of ERK1/2 by RASEF in lung cancer cells. A, expression of MAPK signal molecules and their phosphorylation levels in DMS114 cells transfected with RASEF expression vector or mock plasmid. B, expression of MAPK signal molecules and their phosphorylation levels in NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC). C and D, expression levels of downstream target genes of MAPK cascade were regulated by RASEF expression in lung cancer cells. Total RNA from BEAS-2B and DMS114 cells transfected with RASEF expression vector or mock plasmid (C) and A549 and NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC; D) were subjected to reverse-transcription reaction, followed by PCR reaction to evaluate the expression levels of CCND1, CCNB1, and CDKN1A transcription. Western blotting with antiphosphorylated ERK1/2 antibody was conducted to confirm the change of ERK1/2 phosphorylation according to RASEF expression.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 5. Identification of ERK1/2-interacting sites on RASEF. A, interaction of endogenous RASEF with endogenous ERK1/2. The immunoprecipitates obtained using anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. B, schematic representation of various partial constructs of RASEF expression vector. C and D, determination of the ERK1/2-interacting regions on RASEF by immunoprecipitation experiments using DMS114 cells transfected with vectors expressing partial RASEF protein. COOH-terminal part of RASEF (codons 520–575) was likely to be ERK1/2-interacting region.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection