{"id":608519,"date":"2024-06-07T20:00:00","date_gmt":"2024-06-08T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/exploring-plant-derived-phytochrome-chaperone-proteins-for-light-switchable-transcriptional-regulation-in-mammals-nature-communications\/"},"modified":"2024-06-08T03:01:00","modified_gmt":"2024-06-08T07:01:00","slug":"exploring-plant-derived-phytochrome-chaperone-proteins-for-light-switchable-transcriptional-regulation-in-mammals-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/exploring-plant-derived-phytochrome-chaperone-proteins-for-light-switchable-transcriptional-regulation-in-mammals-nature-communications\/","title":{"rendered":"Exploring plant-derived phytochrome chaperone proteins for light-switchable transcriptional regulation in mammals – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
<\/div>\n

Plasmid construction<\/h3>\n

Comprehensive design and construction details for all expression vectors are provided in Supplementary Data 1<\/a>. The DNA sequences for PTRC components are provided in Supplementary Table 1<\/a>. Some plasmids were constructed using a MultiS One Step Cloning Kit (C113-01, Vazyme) according to the manufacturer\u2019s instructions. All relevant genetic components have been confirmed by sequencing (Personalbio).<\/p>\n

Cell culture and transfection<\/h3>\n

Human embryonic kidney cells (HEK293T cells, CRL-1573, ATCC), baby hamster kidney cells (BHK-21; CCL-10, ATCC), telomerase-immortalized human mesenchymal stem cells (hMSC-TERT; SCRC-4000, ATCC), B16-F10 melanoma cells (CRL-6475, ATCC), HEK293-derived Hana3A cells engineered for constitutive expression of RTP1, RTP2, REEP1 and G\u03b1o\u03bb\u03d553<\/a><\/sup>, human cervical adenocarcinoma cells (HeLa; CCL-2, ATCC), human hepatocellular carcinoma cells (Huh7; TCHu182, Chinese Academy of Sciences), human Retinal Pigment Epithelial cell (ARPE-19; Chinese Academy of Sciences), and the mouse fibroblast NIH-3T3 cell line (CRL-1658, ATCC) were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM; C11995500BT, Gibco) supplemented with 10% (vol\/vol) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (vol\/vol) penicillin\/streptomycin solution (ST488-1\/ST488-2, Beyotime). All cell lines were cultured at 37\u2009\u00b0C in a humidified atmosphere containing 5% CO2<\/sub> and were regularly tested for the absence of mycoplasma and bacterial contamination.<\/p>\n

HEK293T, hMSC-TERT, and Hana3A cells were transfected with an optimized polyethyleneimine (PEI)-based protocol. Unless explicitly indicated, all the cell experiments were performed as follows. Briefly, the cells were plated 6\u2009\u00d7\u2009104<\/sup> cells\/well in a 24-well plate and cultured for 18\u2009hours before transfection. Subsequently, cells were incubated for 6\u2009hours with 50\u2009\u00b5L of PEI and DNA mixture at a mass ratio of 3:1 (PEI, molecular weight 40,000, stock solution 1\u2009mg\/mL in double distilled water; 24765, Polysciences). For the transfection of B16-F10, HeLa, Huh7, ARPE-19, and NIH-3T3 cells, the cells were plated at 5\u2009\u00d7\u2009104<\/sup> cells\/well in a 24-well plate and cultivated for 12\u2009hours at the time of transfection using Lipofectamine 8000 transfection reagent (C0533FT, Beyotime) according to manufacturer\u2019s protocol. For transfection, HEK293T cells were plated at 5\u2009\u00d7\u2009104<\/sup> cells\/well in a 24-well plate and cultivated for 12\u2009hours. At the time of transfection, INVI DNA Transfection Reagent (IV1214, Invigentech) was used according to the manufacturer\u2019s protocol. Detailed transfection mixtures are provided in Supplementary Data 2<\/a> and 3<\/a>.<\/p>\n

Small molecule induction<\/h3>\n

Resveratrol (50\u2009mM; R5010, Sigma-Aldrich) was purchased from Sigma-Aldrich, prepared in 50\u2009mM stock solutions in 100% DMSO, stored at \u221220\u2009\u00b0C, and diluted to different working concentrations using DMEM medium. Stocks (100\u00d7) of ABA (10\u2009mM; 90769, Sigma-Aldrich) and RAPA (10\u2009\u03bcM; A606203, Sangon Biotech) were prepared in 100% ethanol and stored at \u221220\u2009\u00b0C. A 100\u00d7 stock of GZV (1\u2009mM; A173303, Adooq Bioscience), DNV (1\u2009mM; RG7227, Adooq Bioscience), and PCB (500\u2009\u03bcM; P14137, Frontier Scientific) were made in DMSO and stored at \u221220\u2009\u00b0C. These molecules were added to cell cultures, so the final concentration was 1\u00d7 at induction time. For mouse experiments, ABA (200\u2009mg\/kg) and PCB (5\u2009mg\/kg) were intraperitoneally injected after being dissolved into 100\u2009\u03bcL PBS (A600100, Sangon Biotech).<\/p>\n

Light illumination<\/h3>\n

For cell experiments, 24-well plates containing the samples were placed below a custom-designed LED array (4\u2009\u00d7\u20096) emitting blue, red, or far-red light (465\u2009nm, 660\u2009nm, or 730\u2009nm; Shenzhen Bested Opto-electronic, with each LED centered above a single well). The illumination intensity was set to 1\u2009mW\/cm2<\/sup> at 660\u2009nm, 1\u2009mW\/cm2<\/sup> at 730\u2009nm, and 1\u2009mW\/cm2<\/sup> at 465\u2009nm, unless explicitly indicated. This illumination process was carried out after transfection in 37\u2009\u00b0C humidified incubators. Plates not subjected to light treatment were wrapped in aluminum foil immediately after transfection. The light intensity was measured at a wavelength of 465\u2009nm, 660\u2009nm, or 730\u2009nm using an optical power meter (Q8230, Advantest).<\/p>\n

For mouse experiments, the devices and LED were sourced from Shenzhen Kiwi Lighting Co. Ltd. The LED beads had a power rating of 50\u2009mW\/cm2<\/sup>, with available wavelengths of 465\u2009nm or 660\u2009nm. The light angle of the lamp was between 115 and 130\u00b0, which was narrowed to 60\u00b0 upon the addition of a spotlight kit. The mice were exposed to illumination 8\u2009hours post-injection, at an intensity of 5\u2009mW\/cm2<\/sup> (1\u2009minute on, 5\u2009minutes off, alternating) for 450\u2009nm or at 10\u2009mW\/cm2 (1\u2009minute on, 5\u2009minutes off, alternating) for 660\u2009nm, unless explicitly indicated.<\/p>\n

SEAP assay<\/h3>\n

The quantification of human placental SEAP in cell culture medium was conducted as previously reported54<\/a><\/sup>. Briefly, 120\u2009\u03bcL of substrate solution [100\u2009\u03bcL of 2\u00d7assay buffer containing 20\u2009mM homoarginine (1483-01-8, Sangon Biotech), 1\u2009mM MgCl2<\/sub>, 21% (w\/w) diethanolamine (pH 9.8), and 20\u2009\u03bcL of substrate containing 120\u2009mM p-nitro phenyl phosphate (333338-18-4, Sangon Biotech)] was added to 80\u2009\u03bcL heat-inactivated (65\u2009\u00b0C for 30\u2009min) cell culture supernatant, and the light absorbance time course at 405\u2009nm was measured using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04). Unless explicitly indicated, SEAP production was detected 24\u2009hours after transfection.<\/p>\n

Fluorescence imaging<\/h3>\n

Fluorescence imaging of EGFP expression in cells was performed with an inverted fluorescence microscope (Olympus IX71, TH4-200, Olympus) equipped with an Olympus digital camera (Olympus DP71, Olympus) and a 495\/535-nm (blue\/green\/red) excitation\/emission filter set, and images were acquired with 480\u2009nm (excitation) and 535\u2009nm (emission) filters and analyzed using Image-Pro Express C software (version ipp6.0) for EGFP signal.<\/p>\n

Luciferase reporter assay<\/h3>\n

Luciferase activity levels were measured using the Dual-Luciferase assay kit (RG005, Beyotime). Briefly, cell samples were treated with 200\u2009\u03bcL cell lysis buffer per well of a 24-well plate for 5\u2009minutes. Lysis supernatants were collected after centrifuged at 13,800\u2009\u00d7\u2009g<\/i> for 5\u2009minutes. The mixture of 20\u2009\u03bcL lysis supernatants and 20\u2009\u03bcL luciferase substrate was added to the 96-well plate. The luminescence signal was detected using the Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments).<\/p>\n

qPCR analysis<\/h3>\n

Cells were harvested for total RNA isolation using an RNAiso Plus kit (9109, Takara). A total of 1\u2009\u03bcg RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (RR047, Takara). qPCR reactions were performed on the LightCycler 96 real-time PCR instrument (Roche) using the SYBR Premix Ex Taq (RR420, Takara) for detecting each target gene, and the 2-\u0394\u0394Ct<\/sup> method was used to calculate relative gene expression. The gRNA sequences used in this study are listed in Supplementary Table 2<\/a>, and the qPCR primers used in this study are listed in Supplementary Table 3<\/a>.<\/p>\n

Western blot analysis<\/h3>\n

Tissue samples were lysed in RIPA buffer (50\u2009mM Tris-HCl, pH 7.5, 150\u2009mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1\u2009mM EDTA pH 8.0, 1% NP-40) containing 1\u2009mM phenylmethanesulfonyl fluoride (PMSF). The mixture was ground into homogenate on ice, and the supernatant was collected by centrifuging at 16,200\u2009\u00d7\u2009g<\/i> for 15\u2009minutes at 4\u2009\u00b0C. The protein concentration in samples was determined using a bicinchoninic acid assay kit (P0012S, Beyotime). Lysates were mixed with loading buffer and boiled for 10\u2009minutes. An equal amount of proteins (30\u2009\u00b5g) were run on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore MA). The membrane was blocked with 5% nonfat milk in TBST buffer (50\u2009mM Tris, 1.37\u2009mM NaCl, 2.7\u2009mM KCl, 0.05% Tween 20, pH 8.0) for 1\u2009hour at room temperature. The membranes were then incubated with anti-tdTomato primary antibody (1:500; A00682, GenScript) or anti-GAPDH primary antibody (1:1000; AF1186, Beyotime) overnight at 4\u2009\u00b0C. After washing three times with TBST buffer, the membrane was incubated with a secondary antibody (1:5000; Alexa Fluor790 Goat Anti-Rabbit, Jackson ImmunoResearch) for 1\u2009hour at room temperature. After washing three times with TBST buffer, the membrane was visualized using a fluorescent Western blot imaging system (LI-COR Odyssey Clx).<\/p>\n

PTRC-mediated gene expression dynamics<\/h3>\n

To evaluate PTRC-mediated gene expression, HEK293T cells transfected with PTRC-mediated transcriptional regulation system [5\u2009ng pDQ521 (PSV40<\/sub>-Gal4-FHL-pA), 300\u2009ng pDQ362 (PhCMV<\/sub>-\u0394PhyA-2\u00d7ZIM3-pA), and 100\u2009ng pYZ430 (5\u00d7UAS-PTATA<\/sub>-SEAP-pA)] were illuminated with red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>) for different time periods (0-48\u2009hours). SEAP production was quantified after illumination.<\/p>\n

To evaluate exposure time-dependent transgene expression, HEK293T cells were transfected with a PTRC-mediated transcriptional regulation system. Twelve hours later, the transfected cells were illuminated with red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>) for different time periods (0-48\u2009hours). SEAP production was quantified at 60\u2009hours after transfection.<\/p>\n

To evaluate illumination intensity-dependent gene expression, HEK293T cells transfected with a PTRC-mediated transcriptional regulation system were illuminated with red light (660\u2009nm) at different light intensities (0\u20132\u2009mW\/cm2<\/sup>) for 48\u2009hours, and then SEAP production was quantified.<\/p>\n

To evaluate the long-term dynamics of light-responsive transcriptional regulation, the PTRC transcriptional regulation system was introduced into HEK293T cells using the INVI DNA Transfection Reagent. After transfection, the cells were either kept in darkness or exposed to red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>). The production of SEAP was quantified daily before the culture medium was replaced with fresh medium.<\/p>\n

To evaluate the reversibility of the PTRC-mediated transcriptional regulation system, HEK293T cells transfected with this system were divided into two groups six hours after transfection. Cells in the ON-OFF-ON group were maintained in darkness for nine hours to activate transcription, followed by six hours of exposure to red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>) to deactivate it. Subsequently, these cells were exposed to far-red light (730\u2009nm, 1\u2009mW\/cm2<\/sup>) for nine hours to reactivate transcription. Conversely, cells in the OFF-ON-OFF group were initially exposed to red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>) for six hours to deactivate transcription, then maintained in darkness for nine hours to activate it, and finally exposed again to red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>) for six hours to deactivate it. The relative mRNA levels of SEAP<\/i> were quantified by qPCR following changes in the treatment conditions.<\/p>\n

To determine the time required to completely remove the transcriptional activation effect, HEK293T cells transfected with the PTRC-mediated transcriptional regulation system were first maintained in darkness for 12\u2009hours to induce transcriptional activation. Subsequently, they were divided into two groups. Cells in the Dark group continued to be kept in darkness, whereas those in the 660\u2009nm group were exposed to red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>). The relative mRNA levels of SEAP<\/i> were quantified using qPCR after exposure to red light for intervals of 0, 1, 2, 3, 4, 5, 6, and 12\u2009hours.<\/p>\n

Assessment of the activation\/deactivation performance of the PTRCCIP-ABA system<\/h3>\n

HEK293T cells transfected with the PTRCCIP-ABA<\/sub> system [100\u2009ng pWY49 (PhCMV<\/sub>-ABI-Gal4-pA), 100\u2009ng pWY51 (PhCMV<\/sub>-PYL1-FHL-pA), 100\u2009ng pYZ430, and 200\u2009ng pDQ362] were treated with 100\u2009\u00b5M ABA in darkness for 12\u2009hours before being divided into three groups. The first group was treated again with 100\u2009\u00b5M ABA and kept in darkness (Dark + ABA). The second group was maintained in darkness without any additional ABA treatment (Dark – ABA), and the third group was exposed to red light (660\u2009nm, 1\u2009mW\/cm2<\/sup>) without any further ABA treatment (660\u2009nm – ABA). The relative mRNA levels of SEAP<\/i> were quantified using qPCR at 0, 1, 2, and 3\u2009hours.<\/p>\n

Assessment of the activation\/deactivation performance of the PTRCDL<\/sub> system<\/h3>\n

HEK293T cells transfected with the PTRCDL<\/sub> system [100\u2009ng pDQ369 (PhCMV<\/sub>-CIBN-Gal4-pA), 100\u2009ng pDQ522 (PhCMV<\/sub>-CRY2PHR<\/sub>-FHL-pA), 100\u2009ng pYZ450, and 200\u2009ng pDQ362] were exposed to blue light (465\u2009nm, 1\u2009mW\/cm2<\/sup>) for 12\u2009hours before being divided into three groups. The first group continued to be exposed to blue light (465\u2009nm\u2212465\u2009nm), the second group was kept in darkness (465\u2009nm – Dark), and the third group was exposed to red light (465\u2009nm\u2212660\u2009nm). The relative mRNA levels of SEAP<\/i> were quantified using qPCR at intervals of 0, 1, 2, and 3\u2009hours.<\/p>\n

Animals<\/h3>\n

The experimental animals, including six-week-old C57BL\/6 male mice and the transgenetic Cre-tdTomato reporter male mice (Gt (ROSA)26Sor<\/i>tm14(CAG-tdTomato)Hze<\/i><\/sup>) were obtained from the ECNU Laboratory Animal Centre. All the animals were kept on a standard alternating 12-hour light\/12-hour darkness cycle and given a normal chow diet [6% fat and 18% protein (wt\/wt)] and water.<\/p>\n

FHY1\/FHL-mediated DNA recombination in mice<\/h3>\n

For six-week-old C57BL\/6 mice, two milliliters (10% of the body weight in grams) of Ringer\u2019s solution (147\u2009mM NaCl, 4\u2009mM KCl, 1.13\u2009mM CaCl2<\/sub>) containing a total of 125\u2009\u03bcg plasmids encoding DocS\/Coh2-mediated split-Cre recombinase system [50\u2009\u03bcg pDQ532 (PhCMV<\/sub>-DocS-CreC-pA), 50\u2009\u03bcg pDQ533 (PhCMV<\/sub>-CreN-Coh2-pA), and 25\u2009\u03bcg pXY185 (PhCMV<\/sub>–loxp<\/i>-STOP-loxp<\/i>-Luciferase-pA)], or \u0394FHY1\/\u0394FHL-mediated split-Cre recombinase system [50\u2009\u03bcg pDQ683 (PhCMV<\/sub>-\u0394FHY1-CreC-pA), 50\u2009\u03bcg pDQ661 (PhCMV<\/sub>-CreN-\u0394FHL-pA) and 25\u2009\u03bcg pXY185] was hydrodynamically injected via tail vein injection. Negative control mice (Vehicle) were hydrodynamically injected with pXY185. At 24\u2009hours after plasmid injection, luciferase reporter expression was measured using an IVIS Lumina II in vivo imaging system (IVIS, PerkinElmer). For 6-week-old transgenetic Cre-tdTomato reporter mice, two milliliters (10% of the body weight in grams) of Ringer\u2019s solution containing a total of 200\u2009\u03bcg plasmids encoding DocS\/Coh2-mediated split-Cre recombinase system (100\u2009\u03bcg pDQ532, 100\u2009\u03bcg pDQ533), or \u0394FHY1\/\u0394FHL-mediated split-Cre recombinase system (100\u2009\u03bcg pDQ683, 100\u2009\u03bcg pDQ661) was hydrodynamically injected via tail vein injection. Negative control mice (Vehicle) were hydrodynamically injected with pcDNA3.1(+). At seven days after plasmid injection, the mice were sacrificed, and the livers were isolated for fluorescence imaging, qPCR, Western blot, and histological analysis. The tdTomato signal from the isolated liver was detected using an IVIS equipped with tdTomato filter sets. The collected fluorescence emission signals were stored in epi-fluorescence units (radiance efficiency), and the total flux was calculated for ROI. All images were analyzed using the Living Image\u00ae 4.3.1 software.<\/p>\n

PTRC-mediated transcriptional regulation in mice<\/h3>\n

Two milliliters (10% of the body weight in grams) of Ringer\u2019s solution containing 248\u2009\u03bcg plasmids [8\u2009\u03bcg pDQ328 (PhCMV<\/sub>-Gal4-FHL-pA), 160\u2009\u03bcg pDQ362 and 80\u2009\u03bcg pYZ450 (5\u00d7UAS-PTATA<\/sub>-luciferase-pA)] were hydrodynamically injected into mice (male, six weeks old) via tail vein injection. Eight hours after plasmid injection, the mice were intraperitoneally injected with 5\u2009mg\/kg PCB and then exposed to red light (660\u2009nm LED, 5\u2009mW\/cm2<\/sup>, 1\u2009minute on, 5\u2009minutes off, alternating) for 16\u2009hours. Negative control mice (Vehicle) were hydrodynamically injected with pYZ450. At 24\u2009hours after plasmid injection, luciferase reporter expression was measured using an IVIS.<\/p>\n

PTRCdcas<\/sub>-mediated regulation of endogenous gene transcription in mice<\/h3>\n

Two milliliters (10% of the body weight in grams) of Ringer\u2019s solution containing a total of 300\u2009\u03bcg plasmids [100\u2009\u03bcg pYZ561 (PU6<\/sub>-sgRNA1Ascl1<\/sub>::PU6<\/sub>-sgRNA2Ascl1<\/sub>::PhCMV<\/sub>-dCas9-pA), 150\u2009\u03bcg pDQ362 and 50\u2009\u03bcg pDQ455 (PhCMV<\/sub>-MS2-FUS-FHL-pA)] was hydrodynamically injected into mice (male, six weeks old) via tail vein injection. At 8\u2009hours after plasmid injection, the mice were intraperitoneally injected with 5\u2009mg\/kg PCB and then exposed to red light (660\u2009nm LED, 5\u2009mW\/cm2<\/sup>, 1\u2009minute on, 5\u2009minutes off, alternating) for 16\u2009hours. Negative control mice (Vehicle) were hydrodynamically injected with NTsgRNA. At 16\u2009hours following illumination, the mice were sacrificed, and the livers were collected. RNA was extracted for qPCR analysis. The gRNA sequences used in this study are listed in Supplementary Table 2<\/a>, and the qPCR primers used in this study are listed in Supplementary Table 3<\/a>.<\/p>\n

PTRCCIP<\/sub>-mediated transcriptional regulation in mice<\/h3>\n

Two milliliters (10% of the body weight in grams) of Ringer\u2019s solution containing 320\u2009\u03bcg plasmids [80\u2009\u03bcg pWY49 (PhCMV<\/sub>-ABI-Gal4-pA), 40\u2009\u03bcg pWY51 (PhCMV<\/sub>-PYL1-FHL-pA), 40\u2009\u03bcg pYZ450 and 160\u2009\u03bcg pDQ362] were hydrodynamically injected into mice (6 weeks old) via tail vein injection. Eight hours after plasmid injection, the mice were intraperitoneally injected with 5\u2009mg\/kg PCB and 200\u2009mg\/kg ABA55<\/a><\/sup> and then exposed to red light (660\u2009nm LED, 5\u2009mW\/cm2<\/sup>, 1\u2009minute on, 5\u2009minutes off, alternating) for 16\u2009hours. At 24\u2009hours after plasmid injection, luciferase reporter expression was measured using an IVIS.<\/p>\n

PTRCDL<\/sub>-mediated transcriptional regulation in mice<\/h3>\n

Two milliliters (10% of the body weight in grams) of Ringer\u2019s solution containing 320\u2009\u03bcg plasmids [80\u2009\u03bcg pDQ369, 40\u2009\u03bcg pDQ522, 40\u2009\u03bcg pYZ450, and 160\u2009\u03bcg pDQ362] were hydrodynamically injected into mice (6 weeks old) via tail vein injection. Eight hours after plasmid injection, the mice were intraperitoneally injected with 5\u2009mg\/kg PCB and then exposed to red light (660\u2009nm LED, 5\u2009mW\/cm2<\/sup>, 1\u2009minute on, 5\u2009minutes off, alternating) or blue light (465\u2009nm LED, 5\u2009mW\/cm2<\/sup>, 1\u2009minute on, 5\u2009minutes off, alternating) as indicated. At 24\u2009hours after plasmid injection, luciferase reporter expression was measured using an IVIS.<\/p>\n

IVIS imaging<\/h3>\n

For in vivo imaging, each mouse was intraperitoneally injected with luciferin substrate solution (150\u2009mg\/kg; luc001, Shanghai Sciencelight Biology Science & Technology) intraperitoneally. Five minutes after the injection, bioluminescence images of the mice were captured using an IVIS. The bioluminescence images were then analyzed using Living Image software (version 4.3.1).<\/p>\n

Liver histology imaging<\/h3>\n

Fresh livers were washed three times with cold PBS to remove impurities such as blood and then fixed in 4% (w\/v) paraformaldehyde (PFA; 30525-89-4, Sangon Biotech) for 2\u2009hours at 4\u2009\u00b0C. Tissue blocks of ~1\u2009cm3<\/sup> were cut and embedded in an optimum cutting temperature compound (OCT; 03803389, Leica). Five \u00b5m thick liver sections were prepared using Cryostat Microtome (CM1950, Leica) and rinsed with PBS. Finally, samples were counterstained with 4\u2019,6-diamidino-2-phenylindole (DAPI; 28718-90-3, Sigma) for 10\u2009minutes. Endogenous gene tdTomato expression was observed on an inverted fluorescence microscope (DMI8, Leica).<\/p>\n

Ethics<\/h3>\n

The experiments involving animals were approved by the East China Normal University (ECNU) Animal Care and Use Committee and in direct accordance with the Ministry of Science and Technology of the People\u2019s Republic of China on Animal Care guidelines. The protocol (protocol ID: m20220412, m20220506) was approved by the ECNU Animal Care and Use Committee. All animals were euthanized after the experiments were terminated.<\/p>\n

Statistical analysis<\/h3>\n

All in vitro data are expressed as the mean\u2009\u00b1\u2009SD of three independent experiments (n<\/i>\u2009=\u20093). For the animal experiments, each treatment group consisted of randomly selected mice (n<\/i>\u2009=\u20094\u20136). The results are expressed as mean\u2009\u00b1\u2009SEM. Statistical significance was analyzed by the Student\u2019s t<\/i> test. Neither animals nor samples were excluded from the study. Differences were considered statistically significant at p<\/i>\u2009<\u20090.05 (*), very significant at p<\/i>\u2009<\u20090.01 (**), and extremely significant at p<\/i>\u2009<\u20090.001 (***). GraphPad Prism software version 6.0 was used for statistical analysis.<\/p>\n

Reporting summary<\/h3>\n

Further information on research design is available in the Nature Portfolio Reporting Summary<\/a> linked to this article.<\/p>\n