MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells

Hu, Xiaonan; Binter, Maximilian; Hufendiek, Karsten; Tode, Jan; Framme, Carsten; Fuchs, Heiko

doi:10.1371/journal.pone.0278158

Cited by 2 publications

(1 citation statement)

References 45 publications

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“…At about 90% confluence, cell passages were performed. The isolation procedure of hRPE cells was based on our previous study [20]. The anterior segment, lens, and vitreous body were removed.…”

Section: Cell Isolation and Culturementioning

confidence: 99%

Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342

Hu,

Meister,

Tode

et al. 2024

PLoS ONE

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Adeno-associated viral transduction allows the introduction of nucleic fragments into cells and is widely used to modulate gene expressions in vitro and in vivo. It enables the study of genetic functions and disease mechanisms and, more recently, serves as a tool for gene repair. To achieve optimal transduction performance for a given cell type, selecting an appropriate serotype and the number of virus particles per cell, also known as the multiplicity of infection, is critical. Fluorescent proteins are one of the common reporter genes to visualize successfully transduced cells and assess transduction efficiencies. Traditional methods of measuring fluorescence-positive cells are endpoint analysis by flow cytometry or manual counting with a fluorescence microscope. However, the flow cytometry analysis does not allow further measurement in a test run, and manual counting by microscopy is time-consuming. Here, we present a method that repeatedly evaluates transduction efficiencies by adding the DNA-stain Hoechst 33342 during the transduction process combined with a microscope or live-cell imager and microplate image analysis software. The method achieves fast, high-throughput, reproducible, and real-time post-transduction analysis and allows for optimizing transduction parameters and screening for a proper approach.

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Section: Cell Isolation and Culturementioning

confidence: 99%

Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342

Hu,

Meister,

Tode

et al. 2024

PLoS ONE

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Brief research report: Transcriptional blockade of angiotensin converting enzyme 2 modelled in human retinal pigment epithelial cells

Ashander,

Lumsden,

et al. 2024

Front. Drug Discov.

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As a key host protein involved in cellular infection by the severe acute respiratory syndrome coronavirus (SARS-CoV-2), angiotensin converting enzyme (ACE)2 is an ideal target for antiviral drugs. Manipulation of transcription provides opportunity for graduated blockade that preserves physiological functions. We sought to develop a model system for evaluating manipulation of ACE2 gene transcription using human retinal pigment epithelium. Retinal pigment epithelial cell isolates were prepared from human posterior eyecups (n = 11 individual isolates). The cells expressed ACE2 transcript and protein, and expression was not induced by hypoxia mimetic dimethyloxaloylglycine, or inflammatory cytokine IL-1β. ACE2 gene transcription factors were predicted in silico and cross-referenced with the human retinal pigment epithelial cell transcriptome, and five candidate transcription factors were identified: ETS proto-oncogene 1 transcription factor (ETS1), nuclear factor I C (NFIC), nuclear receptor subfamily 2 group C member 1 (NR2C1), TEA domain transcription factor 1 (TEAD1), and zinc finger protein 384 (ZNF384). The candidates were individually targeted in cells by transfection with small interfering (si)RNA. Knockdowns reduced mean cellular expression of all the transcription factors in comparison to expression in cells transfected with control non-targeted siRNA. Mean cellular ACE2 transcript was reduced under the condition of NR2C1 knockdown, but not for ETS1, NFIC, TEAD1, and ZNF384 knockdowns. Our findings build on previous work demonstrating the potential for drugging gene transcription. Importantly, we show the value of human retinal pigment epithelium as a system for evaluating ACE2 transcriptional blockade, a possible approach for treating SARS-CoV-2 infection. Brief Research Report.

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MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells

Cited by 2 publications

References 45 publications

Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342

Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342

Brief research report: Transcriptional blockade of angiotensin converting enzyme 2 modelled in human retinal pigment epithelial cells

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