Journal: Journal of nanobiotechnology

Article Title: Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease.

doi: 10.1186/s12951-022-01318-8

Figure Lengend Snippet: Fig. 3 Gb3 detection in α-GLA knockdown HK-2 cells and human podocytes at baseline and under PEG-CZNPs treatment. A–D HK-2 cell analysis A Representative confocal immunofluorescence microscopy images of Gb3 (green), with DAPI counterstaining (blue), in control (siRNA) and α-GLA deficient HK-2 cells (siGLA), with or without exposure to PEG-CZNPs. B Quantification of the fluorescence intensities in the confocal microscope images using ImageJ software. C Representative TEM images of control (siRNA) and α-GLA deficient HK-2 cells (siGLA), with or without exposure to PEG-CZNPs. D Quantitative analysis of Gb3 vesicles in control (siRNA) and α-GLA deficient HK-2 cells (siGLA), with or without exposure to PEG-CZNPs. E–I Human podocyte analysis E Representative confocal immunofluorescence microscopy images of Gb3 (green), with DAPI counterstaining (blue), in control (siRNA) and α-GLA deficient human podocytes (siGLA) with or without exposure to PEG-CZNPs. F Quantification of the fluorescence intensity of confocal microscope images using ImageJ software. G Representative TEM images of control (siRNA) and α-GLA deficient human podocytes (siGLA) with or without exposure to PEG-CZNPs. H Quantitative analysis of Gb3 vesicles from TEM images. I Quantification of the total Gb3 content using by LC–MS/MS data and the Student’s t test. Data shown are the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus siGLA knockdown alone

Article Snippet: IFA was also performed Using primary antibodies against Gb3 (Amsbio, Cambridge, MA), synaptopodin (Sigma Aldrich) and LC3B (Sigma Aldrich).

Techniques: Knockdown, Cell Analysis, Immunofluorescence, Microscopy, Control, Fluorescence, Software, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of nanobiotechnology

Article Title: Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease.

doi: 10.1186/s12951-022-01318-8

Figure Lengend Snippet: Fig. 12 Schematic illustrations of the PEG-CZNP induced attenuation of kidney injury from FD. This occurs via the enhancement of the autophagy flux combined with the activation of TFEB, restoration of AKT/mTOR signaling, and antioxidant effects. PEG-CZNPs thereby alleviate inflammatory and fibrosis pathways and attenuate Gb3-mediated kidney injury

Article Snippet: IFA was also performed Using primary antibodies against Gb3 (Amsbio, Cambridge, MA), synaptopodin (Sigma Aldrich) and LC3B (Sigma Aldrich).

Techniques: Activation Assay

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 1. Protective effect of D4476 and possible association with autophagy in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 (commercially available stable RPE cell line) and primary mouse RPE cells. Cytotoxicity of ARPE-19 cells treated with (A) varying concentrations of CQ (10–100 μM) alone for 24 h; (B) 100 μM CQ with vehicle or different D4476 concentrations (1–20 μM) for 24 h; (C) vehicle, 100 μM CQ, CQ+D4476 and 10 μM D4476 for different time-points. (D and E) Relative survival rate of ARPE-19 cells treated with casein kinase 1 (CK1) inhibitor, IC261, and selective activin receptor-like kinase (ALK)5 inhibitor, SB525334, instead of D4476 with the same combination of drugs for 24 h and (F) 100 μM CQ with vehicle and with 0.5 μM rapamycin (Rap) for 24 h. Cytotoxicity assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay unless otherwise specified. (G) Phase-contrast and fluorescence photo micrographs of primary mouse RPE cells. After 3 weeks of culture, cells were stained with RPE65, marker protein of RPE, and Na+/K+-ATPase, marker of plasma membrane. Scale bar, 20 μm. (H and I) Relative survival rate of primary mouse RPE cells. Cells were treated with vehicle, 100 μM CQ, CQ+D4476, 10 μM D4476, CQ+IC261, 1 μM IC261, CQ+SB525334, 1 μM SB525334, CQ+Rap, and 0.5 μM Rap for 24 h. Mean ± standard deviation (SD), n = 3; ##P < 0.01 and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: MTT Assay, Fluorescence, Staining, Marker, Clinical Proteomics, Membrane, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 2. Attenuation of chloroquine (CQ)-induced inhibition of autophagy by D4476 in adult retinal pigment epithelial (ARPE)-19 cells. (A) Phase-contrast photo micrograph and (B) confocal images of green fluorescent protein-light chaing 3 (GFP-LC3, green)-transfected cells after immunostaining with anti-lysosomal asso ciated membrane protein (LAMP)-1 (red) antibody. Cells were treated with vehicle, 100 μM CQ alone, 10 μM D4476 alone, or 10 μM D4476 + 100 μM CQ for 6 h. 4ʹ,6-Diamidino-2-phenylindole (DAPI, blue) was used to counterstain nuclei. Scale bar, 10 μm. Quantification of LC3-positive vacuole (C) size, (D) number (green), and (E) number of LC3-positive (green) vacuoles colocalized (yellow) to LAMP-1 (red). One-hundred cells per experimental condition were analyzed using fluo rescence microscopy. (F) Western blot and quantitative analyses of Beclin 1, p62, and LC3 A/B expression in ARPE-19 cell lysates 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM rapamycin (Rap) plus 100 μM CQ, and 0.5 μM Rap alone. α-Tubulin was used as a loading control. Mean ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle; *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or Bafilomycin A1 (BA1) alone-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Transfection, Immunostaining, Membrane, Microscopy, Western Blot, Expressing, Control, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 5. Effect of D4476 on chloroquine (CQ)-induced interaction of Beclin 1 B-cell lymphoma 2 (Bcl-2) homology 3 (BH3) domain with Bcl-2 and cell proliferation- associated signaling in adult retinal pigment epithelial (ARPE)-19 cells. (A) immunoblotted or (B) immunoprecipitated ARPE-19 cells. Cells were immunoprecipitated with anti-mouse IgG, as an isotype control, or anti-Bcl-2 antibody, then immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. (C and D) Densitometric analysis of Immunoblots with Beclin 1 and Bcl-2. Quantitative analysis performed using ImageJ software. (E) Western blot and (F) quantitative densitometric analyses of phosphorylated phosphoinositide 3-kinase (p-PI3K), PI3K, p-AKT, AKT, p-mechanistic target of rapamycin (Rap, mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), and JNK in APRE-19 cells 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM Rap+100 μM CQ, and 0.5 μM Rap alone. Data are means ± standard deviation (SD), n = 3; #P < 0.05 and ##P < 0.01 compared to vehicle and *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Control, Western Blot, Software, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 7. c-Jun N terminal kinase (JNK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival percentage of APRE-19 cells treated with 100 μM CQ alone, 100 μM CQ+10 μM SP600125, and 10 μM SP600125 alone for 24 h. (B) immu noblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated with anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. Bars denote densitometric analysis of Immunoblots. (D) Western blots and (E and F) quantitative densi tometric analyses for phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-JNK, JNK, light chain 3 (LC3) A/B, p62, and Bcl-xL in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 6. p38 Mitogen-activated protein kinase (MAPK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival rate of APRE-19 cells 24 h after treatment with vehicle, 100 μM CQ alone, 100 μM CQ+20 μM SB203580, 20 μM SB203580 alone. (B) immunoblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated using anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 and anti-Bcl-2 antibodies. Bars denote densitometric analyses of Immunoblots. (D) Western blot and (E and F) quantitative densitometric analyses of phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), JNK, light chain 3 (LC3) A/B, p62, and Bcl-2 extra-large (Bcl-xL) in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 8. Schematic representation of D4476 effects on crosstalk between autophagy and apoptosis in chlo roquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. D4476 inhibits CQ-induced increase of mechanistic target of rapamycin (mTOR), c-Jun N terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activities. This inhibitory ef fect alters CQ-mediated interaction between Beclin 1 and B-cell lymphoma 2 (Bcl-2) via the Bcl-2 homol ogy 3 (BH3) domain, resulting in release of Beclin 1 from Bcl-2 and activating autophagy. Beclin 1, light chain 3 (LC3) A/B, and p62 are all associated with autophagosome formation and autophagy flux. D4476 may play an important role in the gateway of intersection between autophagy and apoptosis during CQ-induced toxicity, mitigating damage through reinstating cellular homeostasis.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Oxidative medicine and cellular longevity

Article Title: Olaquindox-Induced Liver Damage Involved the Crosstalk of Oxidative Stress and p53 In Vivo and In Vitro.

doi: 10.1155/2020/8835207

Figure Lengend Snippet: Figure 8: Loss of p53 partially inhibited JNK/p38 pathway and activated autophagy pathway. (a) Western blot analysis for JNK, p38, LC3, and Beclin1 in HCT116 cells. (b) The densitometric analysis of the bands in HCT116 cells were performed via the ImageJ 1.46 software. (c) Western blot analysis for JNK, p38, LC3, and Beclin1 in L02 cells. (d) The densitometric analyses of the bands in L02 cells were performed by the ImageJ 1.46 software. (e) Fluorescence microscopy was used to observe the distribution of autophagosomes in L02 cells after MDC staining (400x). The results represent the means ± SD of three independent experiments. ∗p < 0:05 and ∗∗p < 0:01, compared with the control; #p < 0:05 and ##p < 0:01, compared with p53 normal groups.

Article Snippet: The concentrations of primary antibodies were prepared as follows: rabbit polyclonal antibodies against Beclin1 (1 : 1000), p53 (1 : 1000; Santa Cruz, CA, USA), Bax (1 : 2000), Bcl-2 (1 : 1000), LC3 (1 : 1000) (ProteinTech Group, Inc., Chicago, IL, USA), caspase 3 (1 : 500), caspase 9 (1 : 500; Cell Signaling Technology, Beverly, MA, USA), and PARP (1 : 1000; Beyotime, Haimen, China) and mouse polyclonal antibodies against β-actin (1 : 1000) and Cytochrome C (Cyt C) (1 : 1000, Zhongshan Golden Bridge, Beijing, China).

Techniques: Western Blot, Software, Fluorescence, Microscopy, Staining, Control

Journal: ACS biomaterials science & engineering

Article Title: Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

doi: 10.1021/acsbiomaterials.4c00885

Figure Lengend Snippet: Figure 1. PSC-MATRIX design and summary of properties of PSC-MATRIX investigated. (A) Schematic of the PSC-MATRIX platform. The defined substratum is composed of adhesion peptides derived from vitronectin (GAG Binding Peptide, GBP) and fibronectin (cyclic RGD, cRGD) in addition to the GFP-affinity motif (GFP-TRAP). This customized substratum supports PSC adhesion and facilitates the conversion of a soluble orthogonal ligand, GFP, into an immobilized input competent to activate downstream transgene expression via synNotch signaling. (B) PSC- MATRIX was used with mCherry reporter synNotch-hESCs with activating ligand, GFP, dosed at different time points to demonstrate temporal control of artificial signaling events. Then, PSC-MATRIX was used to selectively functionalize regions of a cell culture plate to enable the activation of synNotch-hESCs in a spatially constrained manner. PSC-MATRIX was also implemented to guide mesendoderm/early peri-gastrulation differentiation and to produce midbrain dopaminergic (mDa) neuron progenitors with WNT transgene expression via artificial signaling by the orthogonal ligand nonfluorescent GFP. Figure created with BioRender.

Article Snippet: To generate the peptide-presenting surfaces, we used combinations of biotinylated adhesion peptides�glycosaminoglycan binding peptide (GBP) (GenScript Express, biotin-Ahx-GKKQRFRHRNRKG), cyclic RGD (cRGD) (Carbosynth, cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEGPEG)), and a GFP capturing motif�GFP-TRAP40,41 which was conjugated to a biotinylated polyethylene glycol (PEG) linker (PEG GFP-TRAP).

Techniques: Derivative Assay, Binding Assay, Expressing, Control, Cell Culture, Activation Assay

Journal: ACS biomaterials science & engineering

Article Title: Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

doi: 10.1021/acsbiomaterials.4c00885

Figure Lengend Snippet: Figure 2. Assessment of synNotch-PSC adhesion and activation on varied substrata. (A) Representative phase contrast and fluorescence microscopy images of GFP-responsive reporter-synNotch H9 hESCs dosed with 5 nM GFP after being plated on various functionalized substrata for 4 days. Cells were plated at high density to simulate PSC-differentiation protocols. Single peptide substrata were composed of 5 μM GBP, 5 μM cRGD, or 0.8 μM GFP-TRAP. Dual peptide surfaces were composed of 5 μM GBP or cRGD and 0.4 μM GFP-TRAP. The tri-peptide substratum was composed of 5 μM GBP, 2.15 μM cRGD, and 0.4 μM GFP-TRAP. (B) Mean mCherry fluorescence intensity of GFP-responsive reporter- synNotch H9 hESCs dosed with 5 nM GFP on different substrata combinations after 4 days. One-way ANOVA with Tukey’s multiple comparisons post hoc. ****p < 0.0001. (C) Mean mCherry fluorescence intensity of GFP-responsive reporter-synNotch H9 hESCs on the tri-peptide surface dosed with 5 nM GFP at various GFP-TRAP concentrations on day 2 of culture. One-way ANOVA with Tukey’s multiple comparisons post hoc: **** p < 0.0001. (D) Mean mCherry fluorescence intensity of GFP-responsive reporter-synNotch H9 hESCs plated on the tri-peptide substratum or a Geltrex-coated surface mixed with GFP-TRAP with and without 5 nM GFP supplementation. Two-way ANOVA with Tukey’s multiple comparisons post hoc: ****p < 0.0001. In all plots, n = 3 replicate wells; error bars indicate SEM. Scale bars = 200 μm.

Article Snippet: To generate the peptide-presenting surfaces, we used combinations of biotinylated adhesion peptides�glycosaminoglycan binding peptide (GBP) (GenScript Express, biotin-Ahx-GKKQRFRHRNRKG), cyclic RGD (cRGD) (Carbosynth, cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEGPEG)), and a GFP capturing motif�GFP-TRAP40,41 which was conjugated to a biotinylated polyethylene glycol (PEG) linker (PEG GFP-TRAP).

Techniques: Activation Assay, Fluorescence, Microscopy

Journal: ACS biomaterials science & engineering

Article Title: Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

doi: 10.1021/acsbiomaterials.4c00885

Figure Lengend Snippet: Figure 5. Demonstration of the capacity of PSC-MATRIX to facilitate both stem cell micropatterning and spatially constrained artificial signaling. (A) Experimental configuration of micropatterned cell culture with the functionalized surface in HUESM-CM differentiation medium with or without GFP. (B) Representative day 2 phase contrast and fluorescence microscopy of inducible WNT3a synNotch-hESCs depicts ligand- dependent synNotch activation via mCherry expression. Scale bars = 500 μm. (C) Quantification of average disc diameter calculated using Leica LAS X software and mean pixel intensity of mCherry fluorescence of each disc calculated using ImageJ software (+GFP n = 22; -GFP n = 18). Unpaired t test for statistical significance: ***p < 0.001, ****p < 0.0001; error bars indicate SEM. (D) Experimental configuration illustrating the functionalization of a 2-panel insert; the left panel was only functionalized with 5 μM GBP and 2.15 μM cRGD whereas the right panel was

Article Snippet: To generate the peptide-presenting surfaces, we used combinations of biotinylated adhesion peptides�glycosaminoglycan binding peptide (GBP) (GenScript Express, biotin-Ahx-GKKQRFRHRNRKG), cyclic RGD (cRGD) (Carbosynth, cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEGPEG)), and a GFP capturing motif�GFP-TRAP40,41 which was conjugated to a biotinylated polyethylene glycol (PEG) linker (PEG GFP-TRAP).

Techniques: Cell Culture, Fluorescence, Microscopy, Activation Assay, Expressing, Software