Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: METTL3 facilitated erastin-induced ferroptosis and enhanced mitochondrial ROS. A Cells were treated with or without 2 μM erastin for 24 h, and the m 6 A/A ratios of mRNA were checked by HPLC/MS/MS. B Dot blot ( left ) and quantitative analysis ( right ) were used to detect the total mRNA m 6 A. C HeLa cells were treated with erastin (2 μM), ferrostatin-1 (1 μM), acetylcysteine (NAC, 2 mM), Z-VAD-FMK (10 μM) and necrosulfonamide (NSA, 5 μM) for 24 h, and the m 6 A/A ratios of mRNA were checked by HPLC/MS/MS. D , E HeLa ( D ) and MDA-MB-231 ( E ) cells were treated with 2 μM erastin for 24 h, and the protein expression was assessed by western blot ( left ) and quantitative analysis ( right ). F , G HeLa ( F ) and MDA-MB-231 ( G ) cells were treated with increasing concentrations erastin for 48 h, and the relative cell viability was detected using Cell Counting Kit-8 kit. H HeLa cells were treated with or without erastin (0.1 μM) for 14 days, and the colonization capability was evaluated ( left ) and analyzed ( right ). I Representative transmission electron microscopy images of WT or METTL3 KD HeLa cells treated with erastin (2 μM, 24 h) ( up ). Yellow arrow represent shrunken mitochondria. The length of mitochondria was measured by image J ( down ). J WT and METTL3 KD HeLa cells were treated with or without erastin (2 μM) for 24 h, and lipid ROS production was assayed by flow cytometry using C11-BODIPY. K HeLa cells were transfected with vector control, METTL3 WT plasmid, or METTL3 DA mutant plasmid for 24 h, and lipid ROS production was assayed by flow cytometry using C11-BODIPY. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: Tandem Mass Spectroscopy, Dot Blot, Expressing, Western Blot, Cell Counting, Transmission Assay, Electron Microscopy, Flow Cytometry, Transfection, Plasmid Preparation, Control, Mutagenesis, Comparison

Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: METTL3 triggered ferroptosis via suppressing SLC7A11 transcription. A The volcano plot showed the mutated genes between WT and METTL3 KD cells. B The overlap between the genes involved in ferroptosis and the mutated genes in METTL3 KD cells. C , D The mRNA expression of SLC7A11, ALOX5 and GLCM in HeLa ( C ) and MDA-MB-231 ( D ) was checked by qRT-PCR. E The protein expression of SLC7A11 in HeLa and MDA-MB-231 cells was checked by western blot analysis. F HeLa and MDA-MB-231 cells were transfected with vector control, METTL3 plasmid, and SLC7A11 plasmid for 24 h, and lipid ROS production was assayed by flow cytometry using C11-BODIPY. G , H HeLa ( G ) and MDA-MB-231 ( H ) HeLa cells were transfected with vector control, METTL3 plasmid, and SLC7A11 plasmid for 24 h, and the relative cell viability was detected using Cell Counting Kit-8 kit after treatment with erastin for 48 h. I m 6 A RIP-qPCR analysis of SLC7A11 mRNA in HeLa and MDA-MB-231 cells. J After treatment with Act-D for the indicated times, the mature mRNA levels of SLC7A11 were checked in WT and METTL3 KD HeLa cells by qRT-PCR. K WT or METTL3 KD HeLa cells were treated with 10 μg/ml CHX for the indicated time periods, and then the protein expression of SLC7A11 was detected by western blot ( left ) and quantitatively analyzed ( right ). L Polysome profiling of WT or METTL3 KD HeLa cells was analyzed. M Analysis of SLC7A11 mRNA in polysome/total for the METTL3 KD cells compared to control cells. N , O The SLC7A11 precursor mRNA expression HeLa and MDA-MB-231cells was checked by qRT-PCR. P , Q The luciferase activity of the SLC7A11 promoter in the F-Luc/R-Luc system was measured using the Dual-Lumi™ II Luciferase Assay Kit. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Control, Flow Cytometry, Cell Counting, Luciferase, Activity Assay, Comparison

Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: METTL3 induced the H3K27 trimethylation of SLC7A11 promoter via suppression of KDM6B. A ChIP-qPCR assay was performed to examine H3K27me3, H3K4me3 and H3K27ac binding to the SLC7A11 promoter in HeLa cells. B After treating HeLa ( left ) and MDA-MB-231 cells ( right ) with 2 μM erastin, ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11. C ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11 after transfecting with siRNAs of KDM6B for 24 h in HeLa WT cells. D ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11 after transfecting with siRNAs of KDM6B for 24 h in HeLa WT and METTL3 KD cells. E ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11 after transfecting with siRNAs of KDM6B for 24 h in MDA-MB-231 sh-NC and sh-METTL3 cells. F SLC7A11 mRNA was detected after transfecting with siRNAs of KDM6B for 24 h in HeLa cells by qRT-PCR. G SLC7A11 and KDM6B proteins were detected after transfecting with siRNAs of KDM6B for 48 h in HeLa cells by western blot ( left ) and quantitatively analyzed ( right ). H H3K27me3 and KDM6B proteins were detected by western blot ( left ) and quantitatively analyzed ( right ) in HeLa WT and METTL3 KD cells. I One m 6 A peak was enriched in KDM6B from m 6 A RIP-seq data. J m 6 A RIP-qPCR analysis of KDM6B mRNA in HeLa WT or METTL3 KD cells. K m 6 A RIP-qPCR analysis of KDM6B mRNA in MDA-MB-231 sh-NC or sh-METTL3 cells. L After treatment with Act-D for the indicated times, the mature mRNA levels of KDM6B were checked in WT and METTL3 KD HeLa cells. M YTHDF2 RIP-qPCR analysis of KDM6B mRNA in WT or METTL3 KD HeLa cells. N KDM6B protein was detected after transfecting with vector or YTHDF2 plasmid for 48 h in HeLa cells by western blot analysis ( right ), and mRNA by qRT-PCR ( left ). O After transfecting with control or YTHDF2 plasmid for 24 h, Act-D was added for the indicated times; furthermore the mature mRNA levels of KDM6B were checked in HeLa cells. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: ChIP-qPCR, Binding Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Control, Comparison

Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: GATA3 was involved in METTL3-regulated transcription of SLC7A11. A The venn diagram showed the overlap of transcription factors of SLC7A11 predicted by PROMO, JASPAR, ChIPBase and ChIP-Atlas, respectively. B Heatmap of SLC7A11 predicted transcription factors. C The expression of GATA3 mRNA was checked by qRT-PCR in HeLa and MDA-MB-231 cells. D The expression of GATA3 protein was checked by western blot analysis in HeLa and MDA-MB-231 cells. E m 6 A RIP-qPCR analysis of GATA3 mRNA in HeLa WT and METTL3 KD cells. F The m 6 A in 5′UTR, CDS and 3′UTR of GATA3 in WT or METTL3 KD HeLa cells was analyzed by m 6 A-RIP-qPCR using fragmented RNA. G HeLa cells were transfected with siRNAs of GATA3, pGL3-SLC7A11-WT promoter and pRL-TK plasmid for 48 h, then the luciferase activity of F-Luc/R-Luc was detected using Dual-Lumi™ II Luciferase Assay Kit. H HeLa cells were transfected with siRNAs of GATA3 for 24 h, and SLC7A11 precursor mRNA expression was checked by qRT-PCR. I HeLa cells were transfected with siRNAs of GATA3 for 48 h, and GATA3 and SLC7A11 protein expression were checked by western blot analysis ( left ) and quantitatively analyzed ( right ). J HeLa cells were transfected with siRNAs of GATA3 for 24 h; they were co-transfected with pGL3-SLC7A11 promoter reporter and pRL-TK for another 24 h. The luciferase activity of F-Luc/R-Luc was detected using Dual-Lumi™ II Luciferase Assay Kit. K The expression of GATA3 mRNA was checked by qRT-PCR in HeLa and MDA-MB-231 cells after treating with erastin (5 µM) for 24 h. L The expression of GATA3 protein was checked by western blot analysis in HeLa and MDA-MB-231 cells after treating with erastin (5 µM) for 24 h. M HeLa cells were transfected with siRNAs of GATA3, and then treated with increasing concentrations of erastin for 48 h, and the relative cell viability was detected using Cell Counting Kit-8 kit. N Schematic representation of the mutated promoter in pGL3-Basic-SLC7A11 reporter. O HeLa cells were co-transfected with siRNAs of GATA3, pGL3-SLC7A11 mutant promoter and pRL-TK plasmid for 24 h, then the luciferase activity of F-Luc/R-Luc was detected using Dual-Lumi™ II Luciferase Assay Kit. P HeLa cells were co-transfected with pGL3-SLC7A11 mutant promoter reporter, pRL-TK and METTL3 plasmid for 24 h, and the luciferase activity of F-Luc/R-Luc was detected using Dual-Lumi™ II Luciferase Assay Kit. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Cell Counting, Mutagenesis, Comparison

Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: METTL3 decreased precursor mRNA stability of GATA3 via m 6 A dependent recruitment of Dis3L2. A After treatment with Act-D for the indicated times, the precursor mRNA levels of GATA3 were checked in WT and METTL3 KD HeLa cells by qRT-PCR. B After treatment with Act-D for the indicated times, the precursor mRNA levels of GATA3 were checked in sh-NC and sh-METTL3 MDA-MB-231 cells by qRT-PCR. C Schematic representation of the pmirGLO-GATA3-3′UTR reporter. D Wild-type or METTL3 KD HeLa cells were transfected with pmirGLO-GATA3-3′UTR reporter for 24 h. The translation outcome was determined as a relative signal of F-luc divided by R-luc, the mRNA abundance was determined by qRT-PCR of and R-luc, and the translation efficiency of GATA3 is defined as the quotient of reporter protein production (F-luc/R-luc) divided by mRNA abundance. E HeLa cells were transfected with pmirGLO-GATA3-3′UTR for 24 h and then further treated with Act-D for the indicated times. The mRNA of F-luc was checked by qRT-PCR. F HeLa cells were transfected with pmirGLO-GATA3-3′UTR-Mut for 24 h, and the relative luciferase activity of F-Luc/R-Luc was measured. G YTHDF2 RIP-qPCR analysis of GATA3 precursor mRNA in HeLa cells. H HeLa cells were transfected with vector or YTHDF2, and GATA3 mRNA expression was measured by qRT-PCR. I HeLa cells were transfected with vector or YTHDF2, and proteins expression were measured by western blot analysis. J Protein lysates of HeLa and MDA-MB-231 cells were immunoprecipitated with YTHDF2 antibodies. The binding between YTHDF2 and Dis3L2 protein was checked by immunoprecipitation. K Dis3L2 RIP-qPCR analysis of GATA3 precursor mRNA in HeLa wild type and METTL3 KD cells. L Dis3L2 RIP-qPCR analysis of GATA3 precursor mRNA in HeLa after transfecting with siRNA of YTHDF2 for 24 h. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Binding Assay, Comparison

Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: YY1 was responsible for the erastin induced-upregulation of METTL3. A The expression of METTL3 mRNA was checked by qRT-PCR in HeLa and MDA-MB-231 cells after treating with erastin (2 µM) for 24 h. B The relative luciferase activity of F-Luc/R-Luc of pGL3-Basic-METTL3 promoter after treating with erastin (2 µM) for 24 h. C Venn diagram showed the overlap of transcription factors of METTL3 predicted by PROMO, JASPAR, ChIPBase and ChIP-Atlas, respectively. D The expression of YY1 mRNA was checked by qRT-PCR in HeLa and MDA-MB-231 cells after treating with erastin (2 µM) for 24 h. E The expression of YY1 protein was checked by western blot analysis in HeLa and MDA-MB-231 cells after treating with erastin (2 µM) for 24 h. F The expression of YY1 mRNA was checked by qRT-PCR in HeLa after transfecting with siRNAs of YY1 for 24 h. G The expression of YY1 protein was checked by western blot analysis in HeLa after transfecting with siRNAs of YY1 for 48 h. H The expression of YY1 protein was checked by western blot analysis ( left ) and quantitatively analyzed ( right ) in HeLa after transfecting with erastin and siRNAs of YY1 for 48 h. I Binding sites of YY1 and primer for ChIP-qPCR to the METTL3 promoter region. J ChIP-qPCR assays to examine YY1 binding to the METTL3 promoter in HeLa cells. K ChIP-qPCR assays to examine YY1 binding to the METTL3 promoter in MDA-MB-231 cells. L Schematic representation of the mutant promoter in pGL3-Basic-METTL3 reporter. M HeLa cells were co-transfected with pGL3-METTL3-WT-Luc, pGL3-METTL3-Mut-Luc, pRL-TK plasmid and si-NC or si-YY1-1 and si-YY1-2 for 48 h. Results were presented as the ratio between the activity of the reporter plasmid and pRL-TK. N Correlation between METTL3 and YY1 in cervical cancer patients (n = 306) from TCGA. O Correlation between METTL3 and YY1 in breast cancer patients (n = 1085) from TCGA. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Western Blot, Binding Assay, ChIP-qPCR, Mutagenesis, Transfection, Plasmid Preparation, Comparison

Journal: Journal of Biomedical Science

Article Title: N6-methyladenosine RNA modification regulates the transcription of SLC7A11 through KDM6B and GATA3 to modulate ferroptosis

doi: 10.1186/s12929-024-01100-y

Figure Lengend Snippet: Targeting m 6 A/METTL3 repressed SLC7A11 expression. A m 6 A RIP-qPCR analysis of KDM6B mRNA in HeLa cells treated with or without STM2457(5 μM) for 24 h. B m 6 A RIP-qPCR analysis of GATA3 precursor mRNA in HeLa cells treated with or without STM2457(5 μM) for 24 h. C The KDM6B, GATA3 and SLC7A11 mRNA expression levels were measured by qRT-PCR in HeLa cells treated with or without STM2457 (5 μM) for 24 h. D The KDM6B, GATA3 and SLC7A11 protein expression levels were measured by western blot analysis in HeLa cells treated with or without STM2457 (5 μM) and erastin (2 μM) for 24 h. E The cell viability was measured after with or without STM2457 (5 μM) and erastin (2 μM) for 24 h. F Schematic representation of positions of m 6 A peaks within KDM6B mRNA and the regions targeted by three gRNAs, respectively. G The KDM6B and SLC7A11 mRNA expression levels in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNAs for 24 h. H The KDM6B and SLC7A11 protein expression levels in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNAs for 24 h. I Schematic representation of positions of m 6 A site within GATA3 precursor mRNA and the regions targeted by three gRNAs, respectively; J The threshold cycle (Ct) of qPCR showing SELECT results for detecting m 6 A site in the potential m 6 A site of GATA3 3′UTR in METTL3 KD HeLa, sh-METTL3 MDA-MB-231 and their corresponding control cells. K The threshold cycle (Ct) of qPCR showing SELECT results for detecting m 6 A site in the potential m 6 A site of GATA3 3′UTR in HeLa cells after transfecting with gRNAs and dCas13b-ALKBH5. L The GATA3 and SLC7A11 mRNA expression levels in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNAs for 24 h. M The GATA3 and SLC7A11 protein expression levels in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNA control or gRNA for 24 h. N HeLa cells were transfected with gRNA control, gRNA for GATA3, and dCas13b-ALKBH5 for 24 h and then further treated with Act-D for the indicated times. The precursor mRNA of GATA3 was checked by qRT-PCR. O HeLa cells were transfected with gRNA control, gRNA for GATA3, gRNA for KDM6B, and dCas13b-ALKBH5 for 24 h, and then further treated with increasing concentration of erastin for 48 h. The cell viability was measures after by Cell Counting Kit-8 kit. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison

Article Snippet: Human lung cancer cell A549, cervical cancer cell HeLa and SiHa, breast cancer MDA-MB-231 were commercially obtained from ATCC and maintained by our laboratory.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Control, Concentration Assay, Cell Counting, Comparison

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Kinetic analysis of immobilized human FcRn and a human IgG1 (mAb1) in solution at pH 6.0 utilizing a medium FcRn density on a switchSENSE biosensor chip. (a) shows the schematic assay configuration allowing affinity and avidity to occur simultaneously. mAb1 was injected in five different concentrations as two-fold dilution series with a highest concentration of 300 nM for hIgg1 Fc WT (b) and 60 nM for hIgg1 Fc YTE (c). Each plot (b,c) shows the measured raw data (gray) and the global fit analysis as solid lines (blue fading). The sensorgram display a monophasic association phase and biphasic dissociation phase reflecting affinity and avidity binding mode. The determined kinetic parameters are described in . Illustration a is created with BioRender.com. Table 2. Summary of the affinity and avidity measurements of immobilized human FcRn and an mAb1 Fc variants in solution using a switchSENSE biosensor chip having a medium ligand density. The kinetic rate parameters are determined from analyzing the sensorgrams shown in . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error. Sample Affinity Avidity k ON (x10 6 M −1 s −1 ) k OFF,AFFINITY (x10 −2 s −1 ) K D,AFFINITY (nM) k OFF,AVIDITY (x10 −2 s −1 ) K D,AVIDITY (nM) hIgG1 Fc WT 7.53 ± 0.38 34.2 ± 1.50 45.4 ± 3.0 4.70 ± 0.30 6.24 ± 0.51 hIgG1 Fc YTE 8.42 ± 0.15 2.60 ± 0.06 3.09 ± 0.09 0.25 ± 0.01 0.29 ± 0.01

Article Snippet: Human FcRn molecules were immobilized on the surface of a switchSENSE multi-purpose chip (MPC-96-2-G1R1-S, Dynamic Biosensors).

Techniques: Injection, Concentration Assay, Binding Assay

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Summary of the affinity and avidity measurements of immobilized human FcRn and an mAb1 Fc variants in solution using a switchSENSE biosensor chip having a medium ligand density. The kinetic rate parameters are determined from analyzing the sensorgrams shown in Figure 2 . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error.

Article Snippet: Human FcRn molecules were immobilized on the surface of a switchSENSE multi-purpose chip (MPC-96-2-G1R1-S, Dynamic Biosensors).

Techniques:

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Kinetic analysis of human FcRn immobilized and a human IgG1 (mAb1) Fc YTE mutant in solution at nine different pH values on switchSENSE biosensor chip. mAb1 Fc YTE was injected in five different concentration as two-fold dilution series with a highest concentration of (a) 60 nM at pH 5.8, (b) 60 nM at pH 6.0, (c) 100 nM at pH 6.2, (d) 150 nM at pH 6.4, (e) 200 nM at pH 6.6, (f) 300 nM at pH 6.8, (g) 400 nM at pH 7.0, (h) 800 nM at pH 7.2 and (i) 800 nM at pH 7.4. Note that the x axis for each sensorgram (a – i) has a different time scale. Each plot (a – i) shows the measured raw data (grey) and the global fit analysis as solid lines (blue fading). For (a – h) the dissociation phase is biphasic characterized by two different dissociation rate constants reflecting the affinity (1:1) and the avidity (2:1) binding mode. The interaction displays a biphasic dissociation curve reflecting affinity and avidity binding where one or two hFcrn are engaged with one mAb1 Fc YTE. The dissociation of mAb1 Fc YTE and hFcrn at pH 7.4 (I) is described by a monophasic fit model reflecting the affinity binding mode (1:1) where one hFcrn is engaged with one mAb1 Fc YTE. Panel (J) shows the applied, exemplary biphasic fit model for FcRn with mAb1 Fc YTE mutant injecting 300 nM at pH 6.0. The adequacy of the fit model is confirmed by the minimal residuals, indicating no significant deviation. The association phase occurred to be monophasic while the dissociation phase is biphasic. The overall dissociation curve is superposition of two exponential time-courses, namely the affinity binding mode (fast dissociation) and the avidity binding mode (slow dissociation). The measured data is shown in blue and the fit in black solid lines, whereas the two deconvoluted exponential time-courses are shown in grey as dashed lines. The contribution of fast and slow dissociation to the overall signal change is shown as Amplitude A fast or A slow . The determined kinetic parameters are described in .

Article Snippet: Human FcRn molecules were immobilized on the surface of a switchSENSE multi-purpose chip (MPC-96-2-G1R1-S, Dynamic Biosensors).

Techniques: Mutagenesis, Injection, Concentration Assay, Binding Assay

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Summary of the affinity and avidity measurements of immobilized human FcRn and mAb1 Fc YTE mutant as solute using a switchSENSE biosensor chip having a medium ligand density. The kinetic rate parameters are determined from analyzing the sensorgrams shown in Figure 3 . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error.

Article Snippet: Human FcRn molecules were immobilized on the surface of a switchSENSE multi-purpose chip (MPC-96-2-G1R1-S, Dynamic Biosensors).

Techniques: Mutagenesis

Journal: Biomicrofluidics

Article Title: Toward a modular, integrated, miniaturized, and portable microfluidic flow control architecture for organs-on-chips applications

doi: 10.1063/5.0074156

Figure Lengend Snippet: Commercially available OoC devices, applications, and microfluidic complexities as shown in and their combinations.

Article Snippet: On-chip micropumps to create recirculation are controlled by external pressurized air or vacuum systems to provide pulsatile flow in these devices. table ft1 table-wrap mode="anchored" t5 TABLE II. caption a7 Company and OoC device name Applied tissue/organ/system Microfluidic complexity Representative image(s) Micronit Microtechnologies B.V. & IMEC Smart multi-electrode array-chip Complex tissue models, heart Single chamber and single channel AlveoliX AX12 Lung Single chamber and double channel PimBio B.V. PIMCELL Gut, 3D cell culture Bi/OND inCHIPit-3C ™ 3D tissues Single chamber and multichannel Emulate Inc. Chip-S1 Lung, brain, kidney, liver, intestine Double chamber and multichannel Hesperos Inc. Human-on-a-chip 2-organ, 3-organ, and 4-organ models Multiple chambers and single channel InSphero Inc. Akura ™ Multi-tissues Multiple chambers and double channel Mimetas B.V. OrganoPlate® 3D tissues and organ models3D co-culture of organotypic cells Multiple chambers and multichannel Nortis ParVivo ™ SynVivo Inc. IMN 2&3 series (linear and radial) Lung, blood-brain barrier, cancer TissUse GmbH HUMIMIC ™ 2-organ and 4-organ models Open in a separate window Commercially available OoC devices, applications, and microfluidic complexities as shown in and their combinations.

Techniques: Cell Culture