Journal: Stem Cell Research & Therapy

Article Title: Prostatic lineage differentiation from human embryonic stem cells through inducible expression of NKX3-1

doi: 10.1186/s13287-024-03886-y

Figure Lengend Snippet: Construction and hindgut differentiation of AAVS1-iNKX3-1 ESC line. ( A ) Schematic diagram of knocking NKX3-1 expressing cassette into AAVS1 site. ( B ) Brightfield and mCherry immunofluorescence results of AAVS1-iNKX3-1 cells. Scale bar = 50 μm (BF: Brightfield). ( C ) Immunofluorescence image of NKX3-1 and mCherry after Doxy induction for 48 h in AAVS1-iNKX3-1 cells, with the control without Doxy induction (CTRL: control, Doxy: Doxycycline). ( D ) Relative mRNA expression of NKX3-1 after Doxy induction for 48 h in AAVS1-iNKX3-1 cells ( n = 3). ( E ) Schematic diagram of hindgut differentiation process (ES: human embryonic stem cell, DE: definitive endoderm cell, HG: hindgut cell). ( F ) Flow cytometry results of CXCR4 at the DE stage, as well as the quantification ( n = 6) (ISO: isotype control). ( G ) Immunofluorescence image of SOX17 and FOXA2 at the DE stage. ( H ) Changes in relative mRNA expression during DE differentiation ( n = 4). ( I ) Immunofluorescence image of CDX2 and FOXA1 at the HG stage. ( J ) Flow cytometry results of CDX2 at the HG stage, as well as the quantification ( n = 6). ( K ) Changes in relative mRNA expression during hindgut differentiation ( n = 4). ** p < 0.01, *** p < 0.001

Article Snippet: The composition of the 3D culture medium is the same as the medium for differentiation from hindgut to urogenital sinus cells or based on prostate organoids culture medium used in previous studies [ , ], supplemented with 2 µg/mL Doxycycline (Doxy) (Sigma-Aldrich, #324385).

Techniques: Expressing, Immunofluorescence, Control, Flow Cytometry

Journal: Stem Cell Research & Therapy

Article Title: Prostatic lineage differentiation from human embryonic stem cells through inducible expression of NKX3-1

doi: 10.1186/s13287-024-03886-y

Figure Lengend Snippet: Differentiation of NKX3-1, AR, and FOXA1 triple-positive UGS cells. ( A ) Schematic diagram of the UGS differentiation from hindgut (HG: hindgut cell, UGS: Urogenital sinus cell, Doxy: Doxycycline). ( B - C ) Western blot results ( B ) (Full-length blots/gels are presented in Supplementary Fig. 4A) and immunofluorescence images ( C ) of AR protein in cells treated with DHT, R1881, and without androgen induction as control during UGS differentiation. The molecular weight of the protein was labeled at right. Scale bar, 50 μm (CTRL: control). ( D - F ) Western blot results ( D ) (Full-length blots/gels are presented in Supplementary Fig. 4B) and immunofluorescence images ( E - F ) of AR, FOXA1 and NKX3-1 proteins in cells treated with ( E ) or without ( F ) Doxy during UGS differentiation. The molecular weight of the protein was labeled at right. Scale bar, 50 μm. ( G ) Statistical analysis of the percentages of positive cells for NKX3-1, AR, and FOXA1 with or without Doxy induction during UGS differentiation ( n = 3). ( H ) Relative mRNA expression levels of NKX3-1 , AR , and FOXA1 with or without Doxy induction during UGS differentiation ( n = 3). ( I ) Changes in relative mRNA expression levels of markers at various stages throughout the differentiation process from ESCs to UGS ( n = 3) (ES: human embryonic stem cell, DE: definitive endoderm cell). ( J ) Immunofluorescence results of CK5, CK8/18, and P63 in UGS cells. Scale bar, 50 μm. ( K - L ) Immunofluorescence images ( K ) and relative mRNA expression level ( L ) of NKX3-1 after adding Doxy induction for 6 days, for 3 days’ treatment followed by 3 days’ withdrawal, and without Doxy induction for 6 days during UGS differentiation ( n = 3). Scale bar, 50 μm. ns, not significant ( p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The composition of the 3D culture medium is the same as the medium for differentiation from hindgut to urogenital sinus cells or based on prostate organoids culture medium used in previous studies [ , ], supplemented with 2 µg/mL Doxycycline (Doxy) (Sigma-Aldrich, #324385).

Techniques: Western Blot, Immunofluorescence, Control, Molecular Weight, Labeling, Expressing

Journal: bioRxiv

Article Title: Pancreatic islets undergo functional and morphological adaptation during development of Barth Syndrome

doi: 10.1101/2024.06.28.601122

Figure Lengend Snippet: (A) ShTaz doxycycline breeding scheme. ( B ) Body weight of Taz -KD and WT at 10 and 50 wo, N (10 wo, WT) = 5, N (10 wo, Taz -KD) = 6, N (50 wo, WT) = 9, N (50 wo, Taz -KD) = 10. Paired analysis of Taz gene expression in heart ( C ) and pancreatic islet ( D ) tissue, N = 3. ( E ) Complete lipid class profile (logarithmic scaling) of pancreatic islets from 20 wo WT and Taz - KD, N = 4. ( F ) Significantly (p < 0.05) altered lipid species in Taz -KD pancreatic islets. Data represent mean ± SEM (indicated by error bars); N numbers indicate number of animals; statistical significance was determined by unpaired Student t test: *p < 0.05, **p < 0.01, ****p < 0.0001. Abbreviations: weeks of age (wo), Tafazzin -Knockdown ( Taz -KD), Wildtype (WT), area under curve (AUC), glucose tolerance test (GTT), lipid class abbreviations can be found in (Supplementary material: Abbreviation of lipids).

Article Snippet: Doxycycline (doxy) in a concentration of 625 mg of doxy/kg was added to the standard rodent chow (A153D70623, Ssniff, Germany) leading to induction of short hairpin RNA (shRNA)-mediated knockdown of Taz , as described previously ( ).

Techniques: Expressing, Knockdown

Journal: bioRxiv

Article Title: Pancreatic islets undergo functional and morphological adaptation during development of Barth Syndrome

doi: 10.1101/2024.06.28.601122

Figure Lengend Snippet: ( A ) Generation of a new mouse (shTaz x Mito-roGFP2-Orp1) model that expresses an shRNA against Taz and the mitochondrial H 2 O 2 sensor roGFP2-Orp1. The mice are lifelong feed with doxy. Figure created with Biorender.com. ( B ) Schematic protocol of redox histology to measure in vivo redox state. Before pancreas isolation, shTaz x Mito-roGFP2-Orp1 mice are perfused with NEM via the cardiovascular system. Isolated pancreas is fixed in PFA und cryocuts are staining against insulin to localize the pancreatic islets. Imaged intensity ratio (excitation: 405/488 nm and emission: 500 – 530 nm) of the mito-roGFP2-Orp1 sensor in pancreatic islets reflects the in vivo redox status. ( C ) Representative ratiometric image (ImageJ Lookup table: “Fire”) of mito-roGFP2-Orp1/WT (left) and mito-roGFP2-Orp1/ Taz -KD (right) pancreatic islets at 20 (top) or 50 wo (bottom). Scale bar: 100 µm. ( D ) Normalized percentage change in ratio of the redox state of the mito-roGFP2-Orp1 sensor in pancreatic islets of 20 (left) and 50 (right) wo mito-roGFP2-Orp1/WT and mito-roGFP2-Orp1/ Taz -KD mice. DTT was used as a reductive control (blue). N (WT, 20 wo) = 7, N ( Taz -KD, 20 wo) = 7, N (DTT, 20 wo) = 8, N (WT, 50 wo) = 4, N ( Taz -KD, 50 wo) = 4, N (DTT, 50 wo) = 6. ( E ) Real-time H 2 O 2 imaging of ex vivo 20 wo mito-roGFP2-Orp1/WT and mito-roGFP2-Orp1/ Taz -KD pancreatic islets. Pancreatic islets were incubated with RPMI 1640 at 5% CO 2 and 37 °C, in presence or absence of 1 µM Auranofin and 100 µM exogenously added H 2 O 2 , N = 5. A schematic figure illustrates the CD7 redox measurement with isolated islets, created with Biorender.com. ( F ) Representative western blot and quantification of Prx3 (left), Cat (middle) and GPX4 (right) normalized to β-actin in pancreatic islets of 20 wo Taz -KD mice, N (Prx3) = 4, N (Cat) = 3, N (GPX4) = 4. Data represent mean ± SEM (indicated by error bars); N and n numbers indicate number of animals and experiments, respectively; statistical significance was determined by unpaired Student t test: *p < 0.05, **p < 0.01. Abbreviations: Mitochondria-redox-sensitive- GFP2-Orp1 (Mito-roGFP2-Orp1), doxycycline (doxy), weeks of age (wo), Tafazzin -Knockdown ( Taz -KD), Wildtype (WT), Dithiothreitol (DTT), glucose (Glu, G), catalase (Cat), peroxiredoxin 3 (Prx3), glutathionperoxidase 4 (GPX4), area under the curve (AUC).

Article Snippet: Doxycycline (doxy) in a concentration of 625 mg of doxy/kg was added to the standard rodent chow (A153D70623, Ssniff, Germany) leading to induction of short hairpin RNA (shRNA)-mediated knockdown of Taz , as described previously ( ).

Techniques: shRNA, In Vivo, Isolation, Staining, Control, Imaging, Ex Vivo, Incubation, Western Blot, Knockdown