abcc5  (Bioss)

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: RNA-seq identifies ABCC5 as a potential key downstream effector of PPARγ in HS. (A) Volcano plot illustrating differentially expressed genes between the WT + HS and PPARγ-OE + HS groups. (B) GO enrichment analysis of differentially expressed genes between the WT + HS and PPARγ-OE + HS groups. (C) KEGG pathway enrichment analysis of DEGs between the WT + HS and PPARγ-OE + HS groups. (D) Heatmap displaying expression changes of ABC transporter family members across the indicated groups. (E) Measurement of cellular free fatty acids and triglycerides in cells under the indicated treatments. (F) RT-qPCR analysis of PPARγ mRNA expression in PPARγ NC + HS and PPARγ OE + HS cells. (G) RT-qPCR analysis of selected ABC transporter genes (ABCC5, ABCB1A, ABCC6, TAP2, ABCA6, ABCB4, ABCC10, ABCA2, ABCG4, ABCA1, ABCA8A, ABCA9, ABCB2, ABCB7, and ABCA3) under the specified conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the PPARγ-NC + HS group (E–G). Statistical comparisons were performed using Student's t-test (F–G) or one-way ANOVA (E).

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: Time-dependent changes in ABCC5 expression in vivo. (A) Representative immunofluorescence images of ABCC5 (green) and DAPI (blue) in cardiac tissues from sham mice and from mice subjected to HS at the indicated time points after injury. (B) Representative immunohistochemical staining of ABCC5 in cardiac tissues from sham and HS-injured mice. (C) RT-qPCR analysis of Leptin mRNA in cardiac tissues after 2.5 h or 3 weeks of heat injury. (D) Representative immunofluorescence images of ABCC5 in cardiac sections from PPARγ-cKO mice after HS). (E–F) Representative immunofluorescence images of PPARγ and ABCC5 in cardiac sections from PPARγ-cKO mice at 3 weeks after HS). Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the sham group (B–C). Statistical comparisons were performed using Student's t-test (B) or one-way ANOVA (C).

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Expressing, In Vivo, Immunofluorescence, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: ABCC5 siRNA abolishes the cardioprotective effects of PPARγ overexpression against HS ​. (A) Luciferase activity in cells co-transfected with ABCC5 wild-type or mutant (Mut1/2/3) reporter plasmids and adenovirus expressing PPARγ. (B) CUT&Tag assay using a PPARγ-specific antibody to detect PPARγ binding to the ABCC5 promoter. (C) RT-qPCR analysis of ABCC5 mRNA in cells transfected with control siRNA or ABCC5 siRNA. (D – F) Cell morphology and viability in cells transfected with ABCC5 siRNA and/or PPARγ overexpression vector under HS conditions. (G – H) Apoptosis levels measured by flow cytometry in cells transfected with ABCC5 siRNA and PPARγ-OE under HS conditions. (I – J) DCFH-DA staining for ROS detection in cells transfected with ABCC5 siRNA and PPARγ-OE under HS conditions. (K – L) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (M) Western blot analysis of PPARγ, ABCC5, ABCC1, Leptin, and β-actin (loading control) in cells treated as follows: PPARγ-NC + HS, PPARγ-OE + HS, and PPARγ-OE + ABCC5 siRNA + HS. Molecular weight markers are shown on the right. (N) Quantification of protein levels normalized to β-actin, corresponding to the blots in (M). Data are presented as mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 versus the indicated control, PPARγ + ABCC5 group (A–B), control siRNA group (C), PPARγ-NC + HS group, PPARγ-OE + HS group, or PPARγ-OE + ABCC5 siRNA + HS group (D–L), or versus the PPARγ-NC + HS group and PPARγ-OE + HS group (M − N). Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing, Binding Assay, Quantitative RT-PCR, Control, Plasmid Preparation, Flow Cytometry, Staining, Membrane, Fluorescence, Western Blot, Molecular Weight

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The PPARγ/ABCC5 pathway alleviates lipid accumulation in HS-injured mice ​. (A – D) Cardiac sections from sham mice and from mice at indicated time points after HS were stained with HE (A) , PSR (B) , Masson's trichrome (C) , or Oil Red O (D) (n = 3 per group). (E) Serum levels of HDL-C and LDL-C in sham mice and in mice 3 weeks after HS (n = 6–7 per group). Error bars represent mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the sham group. Statistical comparisons were performed using Student's t-test.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Staining

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: Rosiglitazone pretreatment alleviates HS-induced myocardial injury via the PPARγ/ABCC5 pathway in HL-1 cells ​. (A – C) Cell viability and morphology in cells treated with different concentrations of rosiglitazone (5 μM, 10 μM, 20 μM, 40 μM) under HS conditions. (D – E) Apoptosis levels in cells treated with different concentrations of rosiglitazone under HS conditions. (F–I) DHE staining (F) and DCFH-DA staining (I) for ROS detection in cells treated with different concentrations of rosiglitazone under HS conditions. (J – K) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (L) RT-qPCR analysis of PPARγ, ABCC5, Leptin, and SREBP-1c in cells treated with different concentrations of rosiglitazone under HS conditions. (M – N) Representative Western blots and quantification of PPARγ, ABCC5, ABCC1, ABCG1, ABCA1, and Leptin in cells treated with different concentrations of rosiglitazone under HS conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the control group or the HS group. Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Staining, Membrane, Fluorescence, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The PPARγ agonist rosiglitazone confers pharmacological protection against HS-induced myocardial dysfunction ​. (A – C) Cell viability and morphology in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (D – E) Apoptosis levels measured by flow cytometry in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (F) LDH release in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (G – H) DCFH-DA staining for ROS detection in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (I – J) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (K) RT-qPCR analysis of PPARγ and CPT1β mRNA in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (L) Representative Western blots and quantification of PPARγ, ABCC5, PGC-1α, and PPARγ in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the control group, the HS group, or the ROSI + HS group as indicated. Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Transfection, Flow Cytometry, Staining, Membrane, Fluorescence, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The proposed scheme describing the signaling pathway of PPARγ/ABCC5-elicted cardioprotective effect against HS.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques:

Journal: bioRxiv

Article Title: BRG1 targeting overcomes ABCC-based multidrug resistance induced by paclitaxel

doi: 10.1101/2025.05.01.651609

Figure Lengend Snippet: (A) mRNA level of SMARCA4 and its truncating deletions correlate with transcription yield of some transmembrane drug carriers in cancer cells according to clinical data. Visual Spreadsheet of UCSC Xena Functional Genomics Explorer compares co-expression of SMARCA4 and some multidrug resistance-relevant ABC genes based on TCGA Pan-Cancer (PANCAN). Samples with log2(norm_value+1) < 10.7 were assigned as SMARCA4 low. (B) Deficiency of BRG1 declines transcription of some ABC genes only in SMARCA4 wild-type cell lines. The impact of siBRG1 72 h after cell transfection on the studied ABC gene transcription in various SMARCA4 genotypes was analysed by real-time PCR. Normalized mRNA level of each gene was assumed as 1 in siCTRL. MDA-MB-231 served as SMARCA4 proficient (BRG1 functional), A549 as SMARCA4 deficient (BRG1 truncated, non-functional) and MCF7 as SMARCA4 fusion (BRG1-CARM1 fusion, non-functional). The difference between siCTRL and siBRG1 was analyzed by Student’s t-test with Welsch correction, and “*” when p<0.05, whereas “ns” when p>0.05. (C) BRG1 is enriched at the promoters of ABCC genes, regardless of their transcriptional dependence on BRG1. BRG1 occurrence at the genomic locations spanning TSS of ABCC3 , ABCC5 and ABCC2 in MDA-MB-231 cells was visualized in USCS Genome Browser and BigWig file was derived from MACS2 peak calling. P indicates cutoff for peak detection, when BRG1 was statistically overrepresented at the considered gene promoters. ABCC2 served as a negative control. (D) BRG1-enriched promoters (± 2 kbp from TSS) represent genes functionally linked to various intracellular processes including protein trafficking to endomembrane system. Ontology of genes characterized by BRG1 occurrence at their promoters (minimum FDR (q-value) cutoff for peak detection set at 0.05) in MDA-MB-231 cells was annotated to biological processes in Panther using statistical overrepresentation test. Genes listed in boxes represent two GO terms (GO:0006891 and GO: 0006622) associated with intracellular vesicle-mediated exchange system of membrane components comprising Golgi apparatus and endolysosomal system. (E) Acquired resistance to paclitaxel changes transcription profile of ABC genes that are functionally linked to multidrug resistance. Normalized gene expression between mapped reads of RNA-Seq datasets from basal (non-resistant; marked as “-“) and paclitaxel-resistant (“Ptx”) cells was generated in CuffLinks using GTEx Gene as a template. Results are shown as a heatmap of normalized gene transcription (FPKM). (F-G) Transient silencing of BRG1 downregulates transcription of a common gene subset, which represent molecular function of transmembrane transporter activity (GO:0022857) in paclitaxel-resistant MDA-MB-251 (MDA-PTX) and A549 (A549-PTX) cell lines. (F) Venn diagram of genes characterized by transcription decline in response to BRG1 silencing (Log2FC < -0.5 ; differential gene expression was generated in CuffDiff using RNA-Seq datasets from BRG1 proficient – siCTRL and deficient – siBRG1 paclitaxel-resistant cell lines) were created using https://bioinformatics.psb.ugent.be/webtools/Venn/ (G) GO terms (molecular function) were assigned to the common gene sets of MDA-PTX and A549-PTX in Panther using statistical overrepresentation test. (H) BRG1 silencing alters transcription of ABC genes, which can contribute to multidrug resistance in cells exposed to several cycles of paclitaxel treatment. Heatmap of differential gene expression presents Log2FC generated in CuffDiff based on RNA-Seq data from BRG1 proficient – siCTRL and deficient – siBRG1 paclitaxel-resistant cells. Bolded genes are characterized by relatively high, unchanged expression or substantial transcription increase caused by paclitaxel. (I) mRNA level of ABC transporters was compared between control and BRG1-deficient samples by real-time PCR. Transcription level was normalized first to housekeeping genes (ACTB, GAPDH and HPRT1) and, then, mRNA level of control was assumed as 1. The difference between two means was tested with Student’s t-test, and statistically significant differences are marked with * when p < 0.05 * and ** when p < 0.01. (J) Effect of transient BRG1 silencing on ABCC3, ABCC5 and ABCC10 protein levels. The lysates of paclitaxel-resistant cells transiently silenced siCTRL and siBRG1 were analyzed by Western Blot. BRG1 was used as a control for silencing efficacy. Histone H3 was used as a loading control.

Article Snippet: SMARCA4 Silencer Select siRNA (s13141), SMARCA2 Silencer Select siRNA (s536647), Lipofectamine RNAiMAX, OptiMem, Dynabeads™ Protein G, UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (#15593031), TRI Reagent™, High-Capacity cDNA Reverse Transcription Kit, BigDye® Terminator v3.1 Cycle Sequencing Kit, Hi-Di formamide, SuperSignal™ West Pico Chemiluminescent Substrate, PageRuler™ Prestained Protein Ladder (#01154870), Pierce™ Protease Inhibitor Tablets (EDTA-free; PIC), Paclitaxel Oregon Green™ 488 conjugate (Flutax-2), Lysotracker™ Deep Red, SlowFade™ Glass Soft-set Antifade Mountant (with DAPI), anti-MRP3 (ABCC3) Polyclonal Antibody (PA5101482), anti-MRP10 (ABCC10) Polyclonal Antibody (PA5101678), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 546 (#A-11010), PowerUp™ SYBR® Green Master Mix, TaqMan™ Universal Master Mix II, TaqMan™ Gene Expression Assays (FAM-MGB/20X) for ACTB (Hs01064292_g1), GAPDH (Hs02786624_g1), HPRT1 (Hs03929096_g1), ABCB1 (Hs00184500_m1), ABCC1 (Hs01561483_m1), ABCC2 (Hs00960489_m1), ABCC3 (Hs00978452_m1), ABCC4 (Hs00988721_m1), ABCC5 (Hs00981089_m1), ABCC10 (Hs01056200_m1), ABCG2 (Hs01053790_m1) were from Thermofisher Scientific (Thermofisher Scientific, Warsaw, Poland).

Techniques: Functional Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Derivative Assay, Negative Control, Membrane, Gene Expression, RNA Sequencing, Generated, Activity Assay, Control, Western Blot

Journal: bioRxiv

Article Title: BRG1 targeting overcomes ABCC-based multidrug resistance induced by paclitaxel

doi: 10.1101/2025.05.01.651609

Figure Lengend Snippet: (A-B) Resistance to paclitaxel changes proportion of BRG1 occurrence at the gene promoters. BRG1 peaks were quantified in the following genomic regions: (A) promoters – regions ± 2 kbp from transcription start site (TSS) and enhancers – derived from UCSC table vistaEnhancers, (B) genes (intergenic regions) – derived from UCSC table gtexGene V8. (C) BRG1 shifts from TSS proximal to distal regions in response to repeated MDA-MB-231 cell exposure to paclitaxel. BRG1 peaks were quantified with respect to the TSS by bedtools ClosestBed, while taking MACS2 peak summits of BRG1 and TSS derived from UCSC table gtexGene V8. Counts were normalized to peak number at – 1 kbp. (D) Gained resistance to paclitaxel is associated with BRG1 spreading at the gene transcription start site. BRG1 occurrence in the region spanning TSS (± 0.5 kbp) was monitored by plotting a profile and heatmap of BRG1 peaks against TSS as a reference point (computeMatrix: plotHeatmap; regions sorted in descending order by mean without clustering). (E) BRG1, EP300 and H3K4me3 are enriched at the promoter of ABCC5 gene regardless of MDA-MB-231 cell resistance to paclitaxel. Genome coverage (as BigWig derived from -bamCoverage) with the two proteins and histone modification around TSS of ABCC5 was visualized in USCS Genome Browser. (F) BRG1 and EP300 are less centered at the transcriptionally active TSS in paclitaxel-resistant cells. The distribution of these two enzymes at H3K4m3 positive TSS in MDA-MB-231 non-versus paclitaxel-resistant was counted (-computeMatrix) and visualized (-plotHeatmap) in the region spanning TSS ± 2.5 kbp. Regions were clustered with respect to H3K4me3 intensity. (G) resistance to paclitaxel declines BRG1 corelation with H3K4me3, but enhances co-occurrence with EP300. BRG1, EP300 and H3K4me3 genomic co-distribution was calculated as Spearman correlation coefficient (-multiBigWigSummary and - plotCorrelation; genomic regions were taken as H3K4me3 peaks that overlap TSS). (H) Paclitaxel resistance of MDA-MB-231 is associated with substantial recruitment of BRG1 to new subset of gene promoters. Gene promoters (TSS ± 2 kbp) characterized by BRG1 peaks in non-resistant and paclitaxel-resistant phenotypes were plotted in venn diagram. Extruded, de novo and remained promoters refer to promoters enriched BRG1 in non-resistant cells only, in paclitaxel-resistant cells, and in both cell phenotypes, respectively. The possible enzyme shift in the considered regions were not taken into consideration. (I) BRG1 is recruited de novo to promoters of genes, which are functionally linked to numerous processes including ER-Golgi-endosome-lysome transport. Gene ontology (GO: biological process complete) of BRG1 de novo enriched promoters in paclitaxel-resistant MDA-MB-231 cells was assigned in Panther using statistical overrepresentation test

Article Snippet: SMARCA4 Silencer Select siRNA (s13141), SMARCA2 Silencer Select siRNA (s536647), Lipofectamine RNAiMAX, OptiMem, Dynabeads™ Protein G, UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (#15593031), TRI Reagent™, High-Capacity cDNA Reverse Transcription Kit, BigDye® Terminator v3.1 Cycle Sequencing Kit, Hi-Di formamide, SuperSignal™ West Pico Chemiluminescent Substrate, PageRuler™ Prestained Protein Ladder (#01154870), Pierce™ Protease Inhibitor Tablets (EDTA-free; PIC), Paclitaxel Oregon Green™ 488 conjugate (Flutax-2), Lysotracker™ Deep Red, SlowFade™ Glass Soft-set Antifade Mountant (with DAPI), anti-MRP3 (ABCC3) Polyclonal Antibody (PA5101482), anti-MRP10 (ABCC10) Polyclonal Antibody (PA5101678), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 546 (#A-11010), PowerUp™ SYBR® Green Master Mix, TaqMan™ Universal Master Mix II, TaqMan™ Gene Expression Assays (FAM-MGB/20X) for ACTB (Hs01064292_g1), GAPDH (Hs02786624_g1), HPRT1 (Hs03929096_g1), ABCB1 (Hs00184500_m1), ABCC1 (Hs01561483_m1), ABCC2 (Hs00960489_m1), ABCC3 (Hs00978452_m1), ABCC4 (Hs00988721_m1), ABCC5 (Hs00981089_m1), ABCC10 (Hs01056200_m1), ABCG2 (Hs01053790_m1) were from Thermofisher Scientific (Thermofisher Scientific, Warsaw, Poland).

Techniques: Derivative Assay, Modification

Journal: bioRxiv

Article Title: BRG1 targeting overcomes ABCC-based multidrug resistance induced by paclitaxel

doi: 10.1101/2025.05.01.651609

Figure Lengend Snippet: (A-D, H-K) The lack of SWI/SNF activity declines transcription and intracellular abundance of ABCC3, ABCC5 and ABCC10 in paclitaxel-resistant phenotypes. mRNA level of ABCC3 , ABCC5 and ABCC10 was compared by real-time PCR in paclitaxel-resistant MDA-MB-231 (A) and A549 (C) cells exposed and not to PFI3 (2.5 uM, 72h) and paclitaxel-resistant MDA-MB-231 (H) and A549 (J) cells exposed and not to ACBI1 (0.5 uM, 72h). Transcription level was normalized first to housekeeping genes ( ACTB , GAPDH and HPRT1 ) and for control sample was assumed as 1. The difference between two means was tested with Student’s t-test, and statistically significant differences are marked with * when p < 0.05 *, ** when p < 0.01, *** when p < 0.001. (B,D) Impact of PFI3 on ABCC10 protein level in paclitaxel-resistant MDA-MB-231 (B) and A549 (D) cell lysates was tested by Western Blot. (I,K) Effect on ACBI1-targeted SWI/SNF subunits degradation on ABCC3 protein level in paclitaxel-resistant MDA-MB-231 (I) and A549 (K) cell lysates was tested by Western Blot. Histone H3 was used as a loading control. (E, L) Expression and localization of ABC transporters was visualized by immunocytostaining followed by confocal microscopy in non-treated vs PFI3 (E) and ACBI1 (L) -targeted cells. Green fluorescence of ABC transporters derived from Alexafluor488-conjugated secondary antibody, blue fluorescence of DNA from DAPI, whereas lysosomal red fluorescence from LysoTracker Deep Red. The fluorescence intensity (F,M) and colocalization (G,N) was determined in arbitrary units (a.u.) with Leica Application Suite X. The difference between two means was tested with Student’s t-test, and statistically significant differences are marked with * when p < 0.05 *, ** when p < 0.01, *** when p < 0.001. (O) Confocal microscopy imaging of lysosomal membrane proteins in control and PFI3-treated MDA-MB-231-PTX cells. ABC transporters were visualized by immunocytostaining followed by confocal microscopy. Red fluorescence of ABC transporters is derived from R-phycoerythrin-labelled secondary antibody and green fluorescence of LAMP1 is derived from Alexafluor488-conjugated secondary antibody. The scans of lysosomes were deconvolved using 3D-Deconwolution accessible in Leica Application Suite X software (LAS X, Leica Microsystems, Germany).

Article Snippet: SMARCA4 Silencer Select siRNA (s13141), SMARCA2 Silencer Select siRNA (s536647), Lipofectamine RNAiMAX, OptiMem, Dynabeads™ Protein G, UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (#15593031), TRI Reagent™, High-Capacity cDNA Reverse Transcription Kit, BigDye® Terminator v3.1 Cycle Sequencing Kit, Hi-Di formamide, SuperSignal™ West Pico Chemiluminescent Substrate, PageRuler™ Prestained Protein Ladder (#01154870), Pierce™ Protease Inhibitor Tablets (EDTA-free; PIC), Paclitaxel Oregon Green™ 488 conjugate (Flutax-2), Lysotracker™ Deep Red, SlowFade™ Glass Soft-set Antifade Mountant (with DAPI), anti-MRP3 (ABCC3) Polyclonal Antibody (PA5101482), anti-MRP10 (ABCC10) Polyclonal Antibody (PA5101678), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 546 (#A-11010), PowerUp™ SYBR® Green Master Mix, TaqMan™ Universal Master Mix II, TaqMan™ Gene Expression Assays (FAM-MGB/20X) for ACTB (Hs01064292_g1), GAPDH (Hs02786624_g1), HPRT1 (Hs03929096_g1), ABCB1 (Hs00184500_m1), ABCC1 (Hs01561483_m1), ABCC2 (Hs00960489_m1), ABCC3 (Hs00978452_m1), ABCC4 (Hs00988721_m1), ABCC5 (Hs00981089_m1), ABCC10 (Hs01056200_m1), ABCG2 (Hs01053790_m1) were from Thermofisher Scientific (Thermofisher Scientific, Warsaw, Poland).

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Control, Western Blot, Expressing, Confocal Microscopy, Fluorescence, Derivative Assay, Imaging, Membrane, Software

Journal: Heliyon

Article Title: CD133 + /ABCC5 + cervical cancer cells exhibit cancer stem cell properties

doi: 10.1016/j.heliyon.2024.e37066

Figure Lengend Snippet: Hela/Taxol cells are more tumor stemness and CD133+ABCC5+ may be a new tumor stem cell marker. (A) Flow assay of ABCC5+, FOXM1+, CD24 + CD44+ABCC5+, CD44+ABCC5+, CD24 + CD44+FOXM1+, CD44+FOXM1+, CD133+ABCC5+, and CD133+FOXM1+ in Hela and Hela/Taxol cells and their statistical plots. (C–D) Sorted cell stem cell spheroid formation assay and statistical bar graph of Hela, Heta/Taxol cells. (E–F) Expression levels of Sox2 and FOXM1 proteins in CD133+ABCC5+ cells sorted from Hela or Hela/Taxol cells and bar graphs. The data are expressed as mean standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After collection and two PBS washes, cells from adherent cultures of Hela and Hela/Taxol were incubated with PE-labeled mouse anti-human CD133 antibody (BD) and Alexa Fluor 488-labeled secondary antibody for ABCC5 (Invitrogen) at 37 °C for 1 h. After a further two washes with chilled PBS, CD133 and ABCC5 expression was analyzed via flow cytometry using the BD FACSCalibur.

Techniques: Marker, Tube Formation Assay, Expressing, Standard Deviation

Journal: Heliyon

Article Title: CD133 + /ABCC5 + cervical cancer cells exhibit cancer stem cell properties

doi: 10.1016/j.heliyon.2024.e37066

Figure Lengend Snippet: Expression level analysis of Sox2 and FOXM1 proteins in CD133+ABCC5+ cells sorted from Hela or Hela/Taxol cells( x ‾ ± s , n = 3).

Article Snippet: After collection and two PBS washes, cells from adherent cultures of Hela and Hela/Taxol were incubated with PE-labeled mouse anti-human CD133 antibody (BD) and Alexa Fluor 488-labeled secondary antibody for ABCC5 (Invitrogen) at 37 °C for 1 h. After a further two washes with chilled PBS, CD133 and ABCC5 expression was analyzed via flow cytometry using the BD FACSCalibur.

Techniques: Expressing