mouse anti-klf4 polyclonal antibody  (Thermo Fisher)

Journal: Respiratory research

Article Title: Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

doi: 10.1186/s12931-022-02263-8

Figure Lengend Snippet: Fig. 5 miR-34a-5p inhibitor reduces hyperoxia-induced KLF4 protein expression in cultured lung epithelial cells. MLE-12 cells were transfected with miR-34a-5p inhibitor (Inh) or negative control (NC) at 20 nM. 24 h after transfection, MLE-12 cells were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 24 h. A–D E2F3, E2F1, CCND1, and KLF4 protein levels were measured by Western blot. Densitometry of bands was normalized using calnexin (CNX) levels. E Transcription levels of Klf4 were measured by RT-qPCR. Data are expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 versus air NC, group, †p < 0.05 versus NC in O2 group, RT-qPCR, quantitative real-time PCR

Article Snippet: Non-specific binding was blocked in PBS with 0.02% Tween-20 and 1% dry milk powder (M17200, Research products international, Mt Prospect, IL, USA) for 15 min at room temperature, and subsequently incubated overnight at 4 °C with an anti-KLF4 goat polyclonal antibody (AF3158, Novus Biologicals, Centennial, CO, USA, 1:200 dilution), an anti-KLF4 rabbit polyclonal antibody (ab129473, Abcam, 1:200 dilution), an anti-Spc rabbit polyclonal antibody (AB3786, Millipore, Burlington, MA, USA, 1:200 dilution), an anti-Spc mouse monoclonal antibody (sc518029, Santa Cruz, 1:50 dilution), an anti-Hopx mouse monoclonal antibody (sc398703, Santa cruz, 1:50), an anti-von Willebrand Factor (vWF) rabbit polyclonal antibody (ab6994, abcam, 1:200), or anti-vimentin rabbit monoclonal antibody (ab92547, abcam, 1:200) as primary antibodies.

Techniques: Expressing, Cell Culture, Transfection, Negative Control, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Respiratory research

Article Title: Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

doi: 10.1186/s12931-022-02263-8

Figure Lengend Snippet: Fig. 6 Klf4 siRNA reduces hyperoxia-induced senescence in cultured lung epithelial cells. MLE-12 cells were transfected with Klf4 siRNA or negative control (NC) at 30nM. At 24 h after transfection, MLE-12 cells were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 24 h. For immunofluorescence, Blue = DAPI, nuclei; Green = nuclear lamin B1. A, B KLF4 protein levels were measured by Western blot. Densitometry of bands was normalized using β-actin levels (n = 9–10). C Transcription levels of Klf4 were measured by RT-qPCR (n = 9). D, E Senescence levels in Air and O2 were measured by the nuclear lamin B1 exclusion using immunofluorescence. The number of nuclear lamin B1 exclusion was counted in two randomly selected HPF in each sample (n = 12). F Transcription levels of p53 and p21 were measured by RT-qPCR (n = 9). G Transcription levels of Cxcl2 and PAI-1 were measured by RT-qPCR (n = 9). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus NC in Air group, †p < 0.05, ††p < 0.01, †††p < 0.001 versus NC in O2 group, RT-qPCR, quantitative real-time PCR

Article Snippet: Non-specific binding was blocked in PBS with 0.02% Tween-20 and 1% dry milk powder (M17200, Research products international, Mt Prospect, IL, USA) for 15 min at room temperature, and subsequently incubated overnight at 4 °C with an anti-KLF4 goat polyclonal antibody (AF3158, Novus Biologicals, Centennial, CO, USA, 1:200 dilution), an anti-KLF4 rabbit polyclonal antibody (ab129473, Abcam, 1:200 dilution), an anti-Spc rabbit polyclonal antibody (AB3786, Millipore, Burlington, MA, USA, 1:200 dilution), an anti-Spc mouse monoclonal antibody (sc518029, Santa Cruz, 1:50 dilution), an anti-Hopx mouse monoclonal antibody (sc398703, Santa cruz, 1:50), an anti-von Willebrand Factor (vWF) rabbit polyclonal antibody (ab6994, abcam, 1:200), or anti-vimentin rabbit monoclonal antibody (ab92547, abcam, 1:200) as primary antibodies.

Techniques: Cell Culture, Transfection, Negative Control, Immunofluorescence, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Respiratory research

Article Title: Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

doi: 10.1186/s12931-022-02263-8

Figure Lengend Snippet: Fig. 7 KLF4 protein expression is increased in the lung of mice exposed to hyperoxia and premature infants requiring mechanical ventilation. Newborn C57BL/6J mice (< 12 h old) were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 3 days and then allowed to recover in the air until pnd 7. A Transcription levels of Klf4 were measured by RT-qPCR. B, C KLF4 protein levels were measured by Western blot. Densitometry of bands was normalized using calnexin (CNX) levels. D, E Double immunofluorescence was performed to detect co-localization of KLF4 with Spc. The number of co-localized cells was counted in three randomly selected HPF in each mouse (n = 3). This was normalized to Dapi+ cells. Bar size: 50 μm. F, G Double immunofluorescence were performed to detect co-localization of KLF4 with Spc in the lungs of premature infants requiring mechanical ventilation. The number of co-localized cells was counted in three randomly selected HPF per sample (n = 3). This was normalized to Dapi + cells. Bar size: 50 μm. Data are expressed as mean ± SEM. ***p < 0.001 versus air group at pnd3

Article Snippet: Non-specific binding was blocked in PBS with 0.02% Tween-20 and 1% dry milk powder (M17200, Research products international, Mt Prospect, IL, USA) for 15 min at room temperature, and subsequently incubated overnight at 4 °C with an anti-KLF4 goat polyclonal antibody (AF3158, Novus Biologicals, Centennial, CO, USA, 1:200 dilution), an anti-KLF4 rabbit polyclonal antibody (ab129473, Abcam, 1:200 dilution), an anti-Spc rabbit polyclonal antibody (AB3786, Millipore, Burlington, MA, USA, 1:200 dilution), an anti-Spc mouse monoclonal antibody (sc518029, Santa Cruz, 1:50 dilution), an anti-Hopx mouse monoclonal antibody (sc398703, Santa cruz, 1:50), an anti-von Willebrand Factor (vWF) rabbit polyclonal antibody (ab6994, abcam, 1:200), or anti-vimentin rabbit monoclonal antibody (ab92547, abcam, 1:200) as primary antibodies.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Respiratory research

Article Title: Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

doi: 10.1186/s12931-022-02263-8

Figure Lengend Snippet: Fig. 8 KLF4 protein expression is increased in AT1, endothelia and mesenchymal cells in the hyperoxia-exposed lung. Newborn C57BL/6J mice (< 12 h old) were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 3 days. A, B Dual immunofluorescence was performed to detect co-localization of KLF4 with Hopx, Von Willebrand Factor (vWF), or vimentin. The number of co-localized cells was counted in three randomly selected HPF in each mouse (n = 3). This was normalized to Dapi+ cells. Bar size: 50 μm. C Schematic figure showing hyperoxia-induced senescence via miR-34a/KLF4/p21 pathway. Data are expressed as mean ± SEM. *p < 0.05, ***p < 0.001 versus air group

Article Snippet: Non-specific binding was blocked in PBS with 0.02% Tween-20 and 1% dry milk powder (M17200, Research products international, Mt Prospect, IL, USA) for 15 min at room temperature, and subsequently incubated overnight at 4 °C with an anti-KLF4 goat polyclonal antibody (AF3158, Novus Biologicals, Centennial, CO, USA, 1:200 dilution), an anti-KLF4 rabbit polyclonal antibody (ab129473, Abcam, 1:200 dilution), an anti-Spc rabbit polyclonal antibody (AB3786, Millipore, Burlington, MA, USA, 1:200 dilution), an anti-Spc mouse monoclonal antibody (sc518029, Santa Cruz, 1:50 dilution), an anti-Hopx mouse monoclonal antibody (sc398703, Santa cruz, 1:50), an anti-von Willebrand Factor (vWF) rabbit polyclonal antibody (ab6994, abcam, 1:200), or anti-vimentin rabbit monoclonal antibody (ab92547, abcam, 1:200) as primary antibodies.

Techniques: Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: Whole mount ISH of e9.5 mouse embryos for A) Klf2 mRNA and B) Klf4 mRNA with closeup (A’, B’) and cryosection (A”, B”) of the AVC (black arrow). C) HCR-FISH on whole mount mouse embryos at e8.5, e9.5, and e10 showing Klf2 mRNA (green) , Klf4 mRNA (red), and nuclei (Hoescht, blue). AVC and OFT are outlined in white. D) Corrected total cell fluorescence (CTCF) analysis of HCR-FISH over time for Klf2 and Klf4 in the AVC and OFT ( Klf2: e8.5 (AVC n=3, OFT, n=3), e9 (AVC n=5, OFT, n=4), e9.5 (AVC n=4, OFT, n=3) ( Klf4: e8.5 (AVC n=3, OFT, n=3), e9 (AVC n=5, OFT, n=4), e9.5 (AVC n=4, OFT, n=3). E) Single-Cell RNAseq of mouse hearts showing total Klf2 mRNA and Klf4 mRNA expression in cardiac cell types at e7.75, e8.25, and e9.25. F) mRNA expression as measured by Grey_Value over distance for Klf2 (n=4) and Klf4 (n=5) in the e9.5 AVC. Distance runs left to right, Atrium to Left Ventricle. G) Immunofluorescence on mouse embryos for KLF4 protein (red) in endocardial cells (CD31, white) of the AVC over time (e8.5, e9.5, e10). Nuclei are shown in blue (Hoescht). KLF4 protein alone is shown with endocardium outlined in white. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, LPM-Lateral Plate Mesoderm, LV-Left Ventricle, OFT-Outflow Tract, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Fluorescence, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: Whole mount ISH of e9.5 mouse embryos for A) Klf2 mRNA and B) Klf4 mRNA with closeup (A’, B’) and cryosection (A”, B”) of the OFT. C) KLF4 protein positive endocardial cells over time as percentage of all endocardial cells in the OFT (e8.5 (n=11), e9 (n=3), e9.5 (n=6), e10 (n=3)) and AVC (e8.5 (n=14), e9 (n=6), e9.5 (n=8), e10 (n=3)). D) mRNA expression as measured by Grey_Value over distance for Klf4 (n=3) in the e9.5 OFT. E) KLF4 protein positive endocardial cells versus distance in the e9.5 OFT (n=3). For D) and E), distance runs left to right, Atrium to Left Ventricle. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: DA-Dorsal Aorta, EC-Endocardial Cell, LV-Left Ventricle, OFT-Outflow Tract, RV-Right Ventricle, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Expressing

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) A model of mechanical forces in an e9.5 mouse heart. Red arrows indicate blood flow/shear stress, grey arrows indicate contractile stress. B) Immunofluorescence on e10 mouse heart sections for KLF4 protein (red) and cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht). Closeup of boxed region in B) of the AVC B’) with and B”) without CD31; white arrows indicate cilia; endocardium is outlined in white. Closeups of endocardial cilia are shown in orange and yellow boxes. C) KLF4 protein positive endocardial cells (dotted line) and ciliated endocardial cells (solid line) versus distance in the AVC at e8.5 (n=3), e9.5 (n=4), and e10 (n=3). D) XY plots for correlation of average plot points from C). E) Violin plot for KLF4 protein immunofluorescent signal intensity (CTCF) in ciliated and nonciliated AVC endocardial cells. F) Endocardial ciliation in the AVC over time as percentage of all endocardial cells in wildtype mice (blue) and Ncx1 −/− mice (red) (e8.5 (WT n=8, Ncx1 −/− n=8), e9.0 (WT n=4, Ncx1 −/− n=4), e9.5 (WT n=7, Ncx1 −/− n=7)). G) Ciliated endocardial cells versus distance in the AVC of Ncx1 −/− mice at e9.5 (n=4). For comparison, WT line is provided in blue. H) Immunofluorescence on e9.5 wildtype and Ncx1 −/− mouse heart sections for KLF4 protein (red) and cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht). Version without CD31 is provided; white arrows indicate cilia; endocardium is outlined in white. Closeups of endocardial cilia are shown in yellow boxes. I) KLF4 protein positive endocardial cells in the AVC over time as percentage of all endocardial cells in wildtype mice (blue) and Ncx1 −/− mice (red) (e8.5 (WT n=10, Ncx1 −/− n=10), e9.0 (WT n=6, Ncx1 −/− n=6), e9.5 (WT n=4, Ncx1 −/− n=4)). J) KLF4 protein positive endocardial cells versus distance in the AVC of Ncx1 −/− mice at e9.5 (n=4). For comparison, WT line is provided in blue. For C) , G) , and J) , distance runs left to right, Atrium to Left Ventricle. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, LA-Left Atrium, LV-Left Ventricle, OFT-Outflow Tract, RA-Right Atrium, RV-Right Ventricle, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Shear, Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) Endocardial ciliation in the OFT over time as percentage of all endocardial cells in wildtype mice (blue) and Ncx1 −/− mice (red) (e8.5 (WT n=7, Ncx1 −/− n=7), e9.0 (WT n=4, Ncx1 −/− n=4), e9.5 (WT n=7, Ncx1 −/− n=7)). B) KLF4 protein positive endocardial cells in the OFT over time as percentage of all endocardial cells in wildtype mice (blue) and Ncx1 −/− mice (red) (e8.5 (WT n=10, Ncx1 −/− n=10), e9.0 (WT n=4, Ncx1 −/− n=4), e9.5 (WT n=5, Ncx1 −/− n=5)). Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: OFT-Outflow Tract, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques:

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) HCR-FISH on whole mount mouse embryos at e9.5 showing Kdr mRNA (red) , Vim mRNA (green), and nuclei (Hoescht, blue). The AVC is outlined in white. B) mRNA expression as measured by Grey_Value over distance for Kdr (blue, n=4), Vim (green, n=4) and Klf4 (red, n=6) in the e9.5 AVC. Distance runs left to right, Atrium to Left Ventricle. C) Cushion cellularization as a percentage of total CD31 positive endocardial cells in the AVC and OFT over time (e8.5 (AVC n=3, OFT, n=3), e9 (AVC n=4, OFT, n=4), e9.5 (AVC n=4, OFT, n=4), e10 (AVC n=4, OFT n=5)). D) SingleCell RNAseq GeneSet Enrichment for EndoMT progression. E) UMAP plot of SingleCell RNAseq EndoMT clusters. F) Immunofluorescence on e9.5 wildtype mouse heart sections for FN protein (red) in endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht). Version without CD31 is provided; the AVC is outlined in white. G) Total Klf4 mRNA expression over EndoMT pseudotime from SingleCell RNAseq . H) Correlation matrix between Klf4 and EndoMT regulators; mRNA expression from SingleNuc RNAseq. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). Data are represented as mean ± SEM. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, LV-Left Ventricle, OFT-Outflow Tract, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) Immunofluorescence on e9.5 wildtype and cilia KO ( Kif3a −/− ) mouse heart sections for cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht). A’) Closeup of boxed AVC region without CD31; the AVC is outlined in white. B) KLF4 protein positive endocardial cells in the AVC over time as percentage of all endocardial cells in wildtype mice (blue) and cilia KO mice (green) (e8.5 (WT n=6, cilia KO n=7), e9.0 (WT n=3, cilia KO n=4), e9.5 (WT n=6, cilia KO n=6)). Immunofluorescence on e9.5 C) wildtype and C’) cilia KO ( Kif3a −/− ) whole mount hearts for KLF4 (red) in endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht); the AVC is outlined in white. D) Klf4 mRNA expression as measured by Grey_Value over distance in the AVC of e9.5 wildtype mice (blue, n=3) and cilia KO mice (green, n=4). E) KLF4 protein positive endocardial cells versus distance in e9.5 AVCs of wildtype mice (blue, n=4) and cilia KO mice (green, n=3). F) H&E stain of an e12.5 Dnah11 −/− A-looped mouse heart. G) Nuclear Fast Red stain of e9.5 wildtype and cilia KO mouse hearts with G’) closeups of boxed regions; cushions outlined in black. H) Cushion cellularization as a percentage of total endocardial cells in the AVC of wildtype (blue, n=4), Dnah11 −/− (grey, n=4), cilia KO (green, n=5) and Ncx1 −/− (red, n=4) hearts. I) Immunofluorescence on e9.5 Dnah11 −/− mouse hearts for KLF4 protein (red) and cilia (ARL13B, green) on AVC endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht). Cilia and nuclei alone are given; white arrows indicate cilia. Closeups of endocardial cilia are shown in yellow box. J) Ciliated endocardial cells and KLF4 protein positive endocardial cells in the AVC as percentage of all endocardial cells in e9.5 wildtype (blue) and Dnah11 −/− (grey) mice (cilia: WT n=6, Dnah11 −/− n=6; KLF4: WT n=6, Dnah11 −/− n=6). For D) and E) , distance runs left to right, Atrium to Left Ventricle. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, LV-Left Ventricle, OFT-Outflow Tract, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Immunofluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) Immunofluorescence on e9.5 wildtype and cilia KO ( Ift20 −/− ) mouse heart sections for cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht). A’) Closeup of boxed AVC region without CD31 signal; the AVC is outlined in white. B) Ciliated endocardial cells in the AVC over time as percentage of all endocardial cells in wildtype (blue), Ift20 −/− (dark green) and Kif3a −/− (light green) (e8.5 (WT n=8, Ift20 −/− n=6, Kif3a −/− n=4), e9.0 (WT n=4, Ift20 −/− n=3, Kif3a −/− n=3), e9.5 (WT n=6, Ift20 −/− n=3, Kif3a −/− n=4)). C) KLF4 protein positive endocardial cells in the AVC over time as percentage of all endocardial cells in Ift20 −/− (dark green) and Kif3a −/− (light green) (e8.5 ( Ift20 −/− n=4, Kif3a −/− n=3), e9.0 ( Ift20 −/− n=2, Kif3a −/− n=2), e9.5 ( Ift20 −/− n=4, Kif3a −/− n=2)). Immunofluorescence on e9.5 D) wildtype and E) cilia KO ( Ift20 −/− ) whole mount hearts for KLF4 (red) in endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht); the AVC is outlined in white. HCR-FISH on whole mount F) wildtype and G) Cilia KO mouse embryos at e9.5 showing Klf2 mRNA (green) , Klf4 mRNA (red), and nuclei (Hoescht, blue). Endocardial regions are outlined in white. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, LV-Left Ventricle, OFT-Outflow Tract, RV-Right Ventricle, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) Ciliated endocardial cells in the OFT over time as percentage of all endocardial cells in wildtype (blue), Ift20 −/− (dark green) and Kif3a −/− (light green) (e8.5 (WT n=8, Ift20 −/− n=4, Kif3a −/− n=4), e9.0 (WT n=4, Ift20 −/− n=2, Kif3a −/− n=2), e9.5 (WT n=6, Ift20 −/− n=4, Kif3a −/− n=3)). B) KLF4 protein positive endocardial cells in the OFT over time as percentage of all endocardial cells in Ift20 −/− (dark green) and Kif3a −/− (light green) (e8.5 ( Ift20 −/− n=3, Kif3a −/− n=2), e9.0 ( Ift20 −/− n=2, Kif3a −/− n=2), e9.5 ( Ift20 −/− n=4, Kif3a −/− n=2)). C) KLF4 protein positive endocardial cells in the OFT over time as percentage of all endocardial cells in wildtype (blue) and cilia KO mice (green) (e8.5 (WT n=4, cilia KO n=4), e9.0 (WT n=3, cilia KO n=3), e9.5 (WT n=6, cilia KO n=6)). D) Klf4 mRNA expression as measured by Grey_Value over distance in the OFT of e9.5 cilia KO mice (green, n=3); wildtype is given for comparison in blue (n=3). E) Number of KLF4 protein positive endocardial cells versus distance in the OFT of e9.5 cilia KO mice (green, n=3); wildtype is given for comparison in blue (n=3). F) Immunofluorescence on e9.5 sections of Ift2 +/− / Ncx1 +/− and Ift20 −/− / Ncx1 −/− hearts for KLF4 (red) in endocardial cells (CD31, white). Nuclei are shown in blue (Hoescht); the AVC is outlined in white. KLF4 protein positive endocardial cells in the e9.5 G) AVC and G’) OFT as percentage of all endocardial cells in Ift2 +/− / Ncx1 +/− (blue, AVC n=3, OFT n=3) and Ift20 −/− / Ncx1 −/− (brown, AVC n=4, OFT n=4). Ift20 −/− (green, AVC n=4, OFT n=4) and Ncx1 −/− (red, AVC n=4, OFT n=5) are given for comparison. H) Immunofluorescence on e9.5 wildtype and cilia KO AVC sections for Fibronectin (red). Nuclei are shown in blue (Hoescht); AVC is outlined in white. I) Endocardial cells with a leading edge of fibronectin as percentage of all endocardial cells in e9.5 AVCs of wildtype (blue, n=4) and cilia KO (green, n=5) mice. J) Ciliated endocardial cells and KLF4 protein positive endocardial cells in the OFT as percentage of all endocardial cells in e9.5 wildtype (blue) and Dnah11 −/− (grey) mice (cilia: WT n=6, Dnah11 −/− n=6; KLF4: WT n=6, Dnah11 −/− n=6). For D) and E), distance runs left to right, Right Ventricle to Dorsal Aorta. Statistics : ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Data are represented as mean ± SEM. Abbreviations: A-Atrium, EC-Endocardial Cell, OFT-Outflow Tract, RV-Right Ventricle, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Expressing, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) SingleNuc RNAseq schema. B) UMAP plot of e9.5 hearts. C) Volcano plot of DEG between wildtype and cilia KO ( Ift20 −/− ); KLF4 targets are highlighted. D) UMAP plot of endocardial cluster from B). E) GeneSet Enrichment for EndoMT progression for EndoMT clusters from D). F) UMAP plot of EndoMT clusters. G) Percent enrichment of EndoMT cell types for wildtype, cilia het ( Ift20 +/− ) and cilia KO ( Ift20 −/− ). Statistically significant p-values are in red. Statistics : ns (p > 0.05), * (p ≤ 0.05). Data are represented as mean ± SEM. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, HE-Hematoendothelium, LV-Left Ventricle, PIM-Pro-inflammatory, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques:

Journal: bioRxiv

Article Title: Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development

doi: 10.1101/2024.05.15.594405

Figure Lengend Snippet: A) Top DEGs between cilia KO ( Ift20−/−) and wildtype hearts over EndoMT clusters. B) Endocardial, EndoMT and mesenchymal gene expression in wildtype, cilia het ( Ift20 +/− ) and cilia KO ( Ift20 −/− ). C) Whole mount HCR-FISH for Kdr (red), Vim (green), Emcn (green) and Vcan (red) in wildtype and cilia KO ( Ift20 −/− ) e9.5 AVCs. Nuclei are shown in blue (Hoescht); the AVC is outlined in white. D) Gene Ontology-Biological Processes lower and higher in cilia KO ( Ift20 −/− ). Cushion relevant terms are highlighted. E) Summary graphic: wildtype hearts selectively lose cilia in areas of highest shear stress, allowing KLF4 reduction and subsequent EndoMT/cushion cellularization. Without cilia present, KLF4 is unable to be regionally turned off and EndoMT/cushion cellularization cannot progress. Statistics : ns (p > 0.05), *** (p ≤ 0.001), **** (p ≤ 0.0001). Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, LV-Left Ventricle, WT-Wildtype.

Article Snippet: Antibodies: Mouse anti-Arl13B (1:200 Neuromab), Rabbit anti-Arl13b (1:200 ProteinTech), Goat anti-KLF4 polyclonal (1:100 R&D Systems), Rat anti-CD31 (1:200 BD Biosciences), Rabbit anti-FN (1:200 Sigma Aldrich), Alexa 488 anti-mouse (1:500, Invitrogen), Alexa 488 anti-rabbit (1:500, Invitrogen), Alexa 594 anti-goat (1:500, Invitrogen), Alexa 647 anti-rat (1:500, Invitrogen), Hoescht 33342 (1:2000, Thermo Fisher), Sheep anti-DIG-AP (1:2000, Roche).

Techniques: Expressing, Shear

Journal: Nature Communications

Article Title: Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs

doi: 10.1038/s41467-021-27586-w

Figure Lengend Snippet: a The Epi-like compartment of RtL-embryoids shows three transcriptionally diverging subpopulations, assessed in UMAP representation. b Comparison to Anterior-, Transition-, and Posterior-Epiblast signatures published by Cheng et al. revealed highest transcriptional similarity with anterior-epiblast cells. c Marker gene expression for Anterior-, Transition-, and Posterior-marker genes. d , f IF staining against pluripotency markers during progression from rosette- to lumen stage. The Epi-like compartment of RtL-embryoids (indicated by dotted lines) displays downregulation of naïve-pluripotency markers KLF4 and ESRRB during progression from rosette to lumen stage. Expression of OTX2 was detected at both, rosette and lumen stage. Scale bars = 50 µm. KLF4/ESRRB/OTX2, red; Phalloidin (Phall)/ PODXL, yellow; DAPI, blue. g Primed-pluripotency marker OCT6 was detected to be weakly expressed in some OTX2 + cells at lumen stage; Lower Panel shows magnification of area indicated in panel above. Scale bars = 50 µm. OTX2, red; OCT6, green; PODXL, yellow; DAPI, blue. h Expression of OCT4 and NANOG was detected in RtL-embryoids throughout the culture period. Scale bars = 50 µm. OCT4, green; NANOG, red; DAPI, blue. i pERK pulses were detected in single Epi-like cells of RtL-embryoids at lumen stage, in addition to a diffuse and weak pERK activity in the ExE-like compartment. Scale bars = 50 µm. OTX2, red; pERK, green; DAPI, blue. White arrow indicates pERK+ Epi-like cell. j Single-cell heatmap of core-, naïve-, and primed- pluripotency markers among cells of the Epi-like Subclusters, revealing predominantly naïve-pluripotency factor expression in subcluster 1, while subcluster 2 displayed downregulation of naïve- and upregulation of primed-pluripotency factor in subcluster 2. Subcluster 3 displayed a PGC-like character. Experiments were repeated independently at least three times with similar results ( d – i ).

Article Snippet: Primary antibodies used and dilutions: Goat-polyclonal anti-CDX2 (Santa Cruz: sc-19478; 1:200), Goat-polyclonal anti-GATA4 (Santa Cruz: sc-1237; 1:400), Goat-polyclonal anti-LEFTY1 (R&D Systems: AF746; 1:200), Rabbit-polyclonal anti-p44/42 MAPK (Erk1/2) (Cell signaling: #9101; 1:100), Mouse-polyclonal anti-OCT6 (Absea: 060204E04; 1:10), Mouse-polyclonal anti-ESRRB (Perseus Proteomics:PP-H6705-00; 1:200), Rabbit-polyclonal anti-NANOG (ReproCell: RCAB002P-F; 1:300), Rabbit-polyclonal anti-EOMES (Abcam: ab23345; 1:400), Goat-polyclonal anti-KLF4 (R&D: AF3158; 1:400), Rat-polyclonal anti-PODXL (R&D: MAB1556; 1:300), Goat-polyclonal anti-OTX2 (R&D: AF1979; 1:400), Rabbit-polyclonal anti-GATA3 (Abcam: ab199428; 1:300), Mouse-polyclonal anti-OCT4 (Santa Cruz: sc-5279; 1:300).

Techniques: Marker, Expressing, Staining, Activity Assay