Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) The predicted protein tertiary structure of CcTRPM . The conserved ankyrin repeat (ANK) domain was indicated in the N-terminal. The conserved six transmembrane domain of ion channels structure were shown as TM1-TM6. ( B ) The mRNA expression of CcTRPM in response to different temperatures of 25°C and 10°C by qRT-PCR. ( C ) Fluorescence detection of Fluo-4 AM after heterologous expression of CcTRPM in mammalian HEK293T cells in response to menthol treatment. The recombinant plasmid was generated by inserting the full ORF sequence of CcTRPM into pcDNA3.1(+)-mCherry plasmid. DMSO treatment and ionomycin treatment were used as negative control and positive control, separately. ( D ) Representative images of Ca 2+ imaging after heterologous expression of CcTRPM in mammalian HEK293T cells in response to different temperature treatment. “CcTRPM +10 °C” means the recombinant plasmid of CcTRPM with pcDNA3.1(+)-mCherry was treated with 10 °C. “CcTRPM +25 °C” denotes the pcDNA3.1(+)-mCherry plasmid with CcTRPM was treated with 25 °C. mCherry and Fluo-4 signal are shown in red and green, respectively. Scale bar is 20 μm. ( E ) The developmental expression pattern of CcTRPM for SF at mRNA level using qRT-PCR. 1st, 2nd, 3rd, 4th, and 5th are the nymphs at the first, second, third, fourth and fifth instar, respectively. A-1d, A-5d, and A-10d are the adults at 1 day, 5 days, and 10 days, separately. ( F ) Representative confocal images of CcTRPM in different developmental stages of SF using FISH. Scale bar is 0.5 mm. BF: the bright field. DAPI: the cell nuclei were stained with DAPI and visualized in blue. CcTRPM-Cy3: CcTRPM signal was labeled with Cy3 and visualized in red. Merge: merged imaging of BF, DAPI, and CcTRPM-Cy3 signals. ( G–H ) Comparison of the nymph cuticle ultrastructure, cuticle thickness, and cuticle chitin content of SF 1st instar treated with dsCcTRPM and dsEGFP at 15 days. ( I ) The transition percent of SF 1st instar nymphs treated with dsEGFP, DMSO, dsCcTRPM, CcTRPM antagonist and dsCcTRPM +menthol at 15 days under 10 °C. For RNAi experiments, summer-form 1st instar nymphs were fed with dsEGFP (500 ng/μL) or dsCcTRPM (500 ng/μL). To mimic RNAi effect, summer-form 1st instar nymphs were fed with 0.1% DMSO or CcTRPM antagonist (20 ng/μL). For the rescue experiment, summer-form 1st instar nymphs were fed with the mixture of dsCcTRPM (500 ng/μL) and menthol (1 mg/mL). Then, counted the number of summer-form and winter-form individuals and calculated the transition percent. ( J ) The phenotypes of SF 1st instar nymphs treated with dsEGFP, dsCcTRPM, CcTRPM antagonist and dsCcTRPM +menthol at 15 days under 10 °C. The data in 2B and 2E are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Scale bar is 0.5 mm. Data in 2 G and 2 H are presented as mean ± SD with three biological replications of three technical replicates for each biological replication. Data in 2I are presented as mean ± SD with nine biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Recombinant, Plasmid Preparation, Generated, Sequencing, Negative Control, Positive Control, Imaging, Staining, Labeling, Comparison, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A–B ) The chemical structure of menthol and TRPM antagonist. ( C–E ) Melting curve for qRT-PCR primers of CcTRPM , Ccβ-actin and CcEF-1 . ( F ) The mRNA expression of CcTRPM in response to different temperatures of 25°C and 10°C at 1 day and 2 days by qRT-PCR. ( G ) Effect of menthol treatment at different concentrations on the mRNA expression of CcTRPM under 10 °C condition. ( H ) Effect of TRPM antagonist treatment at different concentrations on the mRNA expression of CcTRPM under 10 °C condition. ( I ) Representative images of Ca 2+ imaging after heterologous expression of CcTRPM in mammalian HEK293T cells in response to menthol treatment. ‘CcTRPM +menthol’ means the recombinant plasmid of CcTRPM with pcDNA3.1(+)-mCherry was treated with menthol. “CcTRPM +DMSO” denotes the pcDNA3.1(+)-mCherry plasmid with CcTRPM was treated with DMSO. mCherry and Fluo-4 signal are shown in red and green, respectively. Scale bar is 20 μm. Data are shown as mean ± SD with three biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Quantitative RT-PCR, Expressing, Imaging, Recombinant, Plasmid Preparation, Comparison, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) The putative miR-252 binding sites in CcTRPM 3’UTR were predicted by miRanda and Targetscan. ( B ) The expression profiles of miR-252 in response to different temperatures of 25°C and 10°C by qRT-PCR. ( C ) In vitro validation of the target interactions between miR-252 and CcTRPM by dual luciferase reporter assays. ( D ) In vivo demonstration of miR-252 targeting CcTRPM by RNA-binding protein immunoprecipitation (RIP) assay. ( E ) Representative confocal images of miR-252 and CcTRPM in 1st, 3rd and 5th instar of SF. Scale bar is 0.5 mm. The signals of DAPI and CcTRPM -Cy3 are same as the above describing. miR-252-FAM: the miR-252 signal was labeled with FAM and visualized in green. Merge: merged imaging of co-localization of cell nucleus, CcTRPM and miR-252. ( F ) Effect of miR-252 agomir and antagomir treatment 3 days on the expression of CcTRPM at mRNA level. The data in 3B and 3 F are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Data in 3 C are presented as mean ± SD with nine biological replications. Data in 3D are presented as mean ± SD with two biological replications of three technical replicates for each biological replication. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, In Vitro, Biomarker Discovery, Luciferase, In Vivo, RNA Binding Assay, Immunoprecipitation, Labeling, Imaging, Comparison, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) Effect of miR-252 agomir treatment on the mRNA expression of miR-252 after 3 days and 6 days under 10 °C. agomir-NC treatment was used as the control. ( B ) The mRNA expression of miR-252 after dsCcTRPM treatment 3 days and 6 days compare with the dsEGFP treatment under 10 °C. ( C–F ) Comparison of the nymph cuticle thickness, cuticle ultrastructure, cuticle chitin staining with WGA-FITC, and chitin content of SF 1st instar after treatment with agomir-NC and agomir-252 at 15 days. Scale bar in (D) is 1μm and in (E) is 100 μm. The two-way arrow indicated the cuticle thickness. The DAPI and WAG-FITC signals were same as the above describing. ( G ) The transition percent of SF 1st instar nymphs treated with agomir-NC, agomir-252, dsEGFP, dsCcTRPM, and dsCcTRPM +antagomir-252 at 15 day under 10 °C. ( H ) The phenotypes of SF 1st instar nymphs treated with agomir-NC, agomir-252, dsCcTRPM, and dsCcTRPM +antagomir-252 at 15 day under 10 °C. Scale bar is 0.5 mm. The data in 4A and 4B are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Data in 4C and 4F are presented as mean ± SD with three biological replications of three technical replications for each biological replication. Data in 4G are presented as mean ± SD with nine biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Expressing, Control, Comparison, Staining, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A–B ) Melting curve for qRT-PCR primers of U6 and miR-252. ( C ) Effect of agomir-252 treatment 2 day and 4 day on the mRNA expression of CcTRPM under 25 °C condition. ( D ) The phenotype of SF 1st instar nymphs treated with agomir-NC and agomir-252 at 10 day at 25 °C. Scale bar is 0.5 mm. Data are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001).

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) Diagram of de novo biosynthesis of insect chitin. Tre: trehalase. HK: hexokinase. G6PI: glucose-6-phosphate isomerase. GFAT: fructose-6-phosphate aminotransferase. GNA: glucosamine-6-phosphate acetyltransferase. AGM: N-acetylglucosamine phosphate mutase. UAP: UDP- N-acetylglucosamine pyrophosphorylase. CHS: chitin synthase. ( B ) A heat map was constructed from the expression levels of chitin biosynthesis enzyme transcripts in C. chinensis after 25°C and 10°C treatment at 3 d, 6 d, and 10 d. ( C ) Effect of RNAi knockdown of 14 transcripts covering all the enzymes in the chitin biosynthesis pathway on the transition percent of SF 1st instar nymphs under 10 °C. ( D–F ) Comparison of the nymph cuticle thickness, cuticle chitin content, and cuticle chitin staining with WGA-FITC of SF 1st instar after treatment with dsEGFP, dsCcTre1, and dsCcCHS1 at 15 d. The WAG-FITC signal was same as the above describing. Scale bar in (F) is 100 μm and 0.5 mm, respectively. ( G–H ) Effect of dsCcTRPM and agomir-252 treatments on the mRNA expression of CcTre1 at 3 day and 6 day under 10 °C. ( I–J ) Effect of dsCcTRPM and agomir-252 treatments on the mRNA expression of CcCHS1 at 3 day and 6 day under 10 °C. The data in ( C ) are shown as the mean ± SD with six independent biological replications of at least 30 nymphs for each biological replication. Data in ( D and E ) are presented as mean ± SD with three biological replications of three technical replications for each biological replication. Data in ( G-J ) are presented as mean ± SD with three biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by * (p<0.05), ** (p<0.01), and *** (p<0.001).

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Construct, Expressing, Knockdown, Comparison, Staining

Journal: Journal of molecular biology

Article Title: Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.

doi: 10.1016/j.jmb.2015.08.006

Figure Lengend Snippet: Fig. 1. Structure and activity of APOBEC3B constructs. (A) Align- ment between parts of A3G and A3B sequences is shown. The sec- ondary structural elements within A3G [21] are shown as boxes above the sequences. The amino-terminal ends of 193-CTD, 195-CTD and 187-CTD are indicated by arrows. (B) Results of KanR reversion assay for 195-CTDintheabsenceofIPTG in the host BH214. The horizontal lines within the data points represent the median values. (C) KanR revertants due to 195-CTD following induction of its transcription using 50 μM or 100 μM IPTG. ** represents P value of b0.01. (D) The results of KanR

Article Snippet: The full length of APOBEC3B was amplified from the human APOBEC3B cDNA purchased from OriGene Technologies (Rockville, MD) and cloned into vector plasmid pSU24 as HindIII-KpnI fragments.

Techniques: Activity Assay, Construct

Journal: Journal of molecular biology

Article Title: Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.

doi: 10.1016/j.jmb.2015.08.006

Figure Lengend Snippet: Fig. 2. KanR reversion and RifR mutation assay for APOBEC3B mutants. (A) Effect of D194A mutation on KanR

Article Snippet: The full length of APOBEC3B was amplified from the human APOBEC3B cDNA purchased from OriGene Technologies (Rockville, MD) and cloned into vector plasmid pSU24 as HindIII-KpnI fragments.

Techniques: Mutagenesis

Journal: Journal of molecular biology

Article Title: Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.

doi: 10.1016/j.jmb.2015.08.006

Figure Lengend Snippet: Fig. 4. Preliminary characterization of purified APOBEC3B 193-CTD. (A) Stability of 193-CTD during reaction incubations. The ability of 193-CTD protein to convert cytosine into uracil is quantified following pre-incubations for different lengths of time. (B) Ability of 193-CTD to turn over. The amount of product generated by the enzyme is shown under two different ratios of substrate to enzyme.

Article Snippet: The full length of APOBEC3B was amplified from the human APOBEC3B cDNA purchased from OriGene Technologies (Rockville, MD) and cloned into vector plasmid pSU24 as HindIII-KpnI fragments.

Techniques: Purification, Generated

Journal: Journal of molecular biology

Article Title: Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.

doi: 10.1016/j.jmb.2015.08.006

Figure Lengend Snippet: Fig. 5. Inability of APOBEC3B NTD to deaminate cytosines. (A) KanR reversion assay for A3B NTD. The revertant frequencies due to the NTD are compared to full-length A3B and the empty vector. (B) Lack of cytosine deamination activity for partially purified NTD. The TCGA-40 substrate (2 pmol) was incubated with different amounts of the NTD alone (lanes 4, 6 and 8, respectively) or together with 10 pmol of 193-CTD (lanes 5, 7 and 9).

Article Snippet: The full length of APOBEC3B was amplified from the human APOBEC3B cDNA purchased from OriGene Technologies (Rockville, MD) and cloned into vector plasmid pSU24 as HindIII-KpnI fragments.

Techniques: Plasmid Preparation, Activity Assay, Purification, Incubation

Journal: Journal of molecular biology

Article Title: Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.

doi: 10.1016/j.jmb.2015.08.006

Figure Lengend Snippet: Fig. 6. Ability of APOBEC3B to deaminate 5mCs. (A) Comparison of KanR revertant frequencies due to conversion of 5mC into T. The abilities of 193-CTD, 187-CTD and full-length A3B to deaminate 5mC are compared with empty vector and A3A. ** represents P value of b0.01 and *** represents P value of b0.001. (B) Comparison of kinetics of cytosine and 5mC deamination by 193-CTD. DNA oligomers containing a single C or 5mC in the indicated sequence context were treated with increasing concentrations of purified 193-CTD and the products were quantified. (C) Kinetics of cytosine and 5mC deamination by partially purified A3A.

Article Snippet: The full length of APOBEC3B was amplified from the human APOBEC3B cDNA purchased from OriGene Technologies (Rockville, MD) and cloned into vector plasmid pSU24 as HindIII-KpnI fragments.

Techniques: Comparison, Plasmid Preparation, Sequencing, Purification

Journal: Journal of molecular biology

Article Title: Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.

doi: 10.1016/j.jmb.2015.08.006

Figure Lengend Snippet: Fig. 7. Sequence alignment of APOBEC3B CTD and APOBEC3 proteins with known structures. Clustal Omega alignment of APOBEC3 single-domain proteins (A3A and A3C) and CTD of two-domain proteins (A3F and A3G) with A3B CTD are shown. The structural motifs are shown according the structure of A3G CTD [21]. The conserved methionine at the junction of NTD and CTD is marked with a green arrow. All the conserved residues between the five proteins are shown in blue and marked by asterisks. The residues that line the hydrophobic pocket in which the conserved methionine resides are shown in red and are marked with a dot.

Article Snippet: The full length of APOBEC3B was amplified from the human APOBEC3B cDNA purchased from OriGene Technologies (Rockville, MD) and cloned into vector plasmid pSU24 as HindIII-KpnI fragments.

Techniques: Sequencing

Journal: Discover Oncology

Article Title: Novel N 6 -methyladenosine (m 6 A) writer METTL16 promotes the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis

doi: 10.1007/s12672-026-04403-8

Figure Lengend Snippet: FTH1 acted as the target of METTL16. A The immunofluorescence assay showed the subcellular localization of METTL16 and FTH1. B The binding motif sites of METTL16 for its target genes (GGAAC). C The FTH1 level in cervical cancer cells was test by the RT-PCR. D RNA immunoprecipitation (RIP) assay was conducted for the binding within METTL16 and FTH1 in HeLa cells. E RIP assay using anti-m 6 A conducted for the m 6 A modification on FTH1 mRNA. * p < 0.05, ** p < 0.01

Article Snippet: Cervical cancer cells were lysed by immunoprecipitation buffer (protease, phosphatase inhibitors, MCE) containing magnetic beads conjugated to specific antibodies (anti-IGF2BP2, anti-METTL16, anti-m 6 A).

Techniques: Immunofluorescence, Binding Assay, Reverse Transcription Polymerase Chain Reaction, RNA Immunoprecipitation, Modification

Journal: Discover Oncology

Article Title: Novel N 6 -methyladenosine (m 6 A) writer METTL16 promotes the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis

doi: 10.1007/s12672-026-04403-8

Figure Lengend Snippet: IGF2BP2 promoted the stability of FTH1 mRNA. A The immunofluorescent staining showed the subcellular localization of IGF2BP2 and FTH1 in HeLa cells. B The positive correlation within IGF2BP2 and FTH1 in clinical samples ( http://gepia.cancer-pku.cn/index.html ). C RNA immunoprecipitation (RIP) assay showed the immunoprecipitated FTH1 mRNA by anti-IGF2BP2 antibody. D RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with IGF2BP2 overexpression. E RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with METTL16 silencing (sh-METTL16-1, sh-METTL16-2). F RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with IGF2BP2 silencing (si-IGF2BP2) and METTL16 overexpression. * p < 0.05, ** p < 0.01

Article Snippet: Cervical cancer cells were lysed by immunoprecipitation buffer (protease, phosphatase inhibitors, MCE) containing magnetic beads conjugated to specific antibodies (anti-IGF2BP2, anti-METTL16, anti-m 6 A).

Techniques: Staining, RNA Immunoprecipitation, Immunoprecipitation, Over Expression

Journal: bioRxiv

Article Title: XIST Drives X-Chromosome Inactivation and Safeguards Female Extraembryonic Cells in Humans

doi: 10.1101/2025.11.19.689206

Figure Lengend Snippet: A. Schematic illustration showing wild type (WT) and XIST inducible knock down (iKD) H9 naive hESCs under trophoblast differentiation medium with or without Doxycycline (Dox) and assaying cell identity and X-chromosome inactivation using microscopy, scRNA-seq, bulk RNA-seq and flow cytometry. B. Representative bright-field microscopy images showing the morphology of H9 XIST iKD clone-1 hESCs at Day 15 of culture in trophoblast differentiation medium with or without Dox. Note: Feeders only are seen in the +Dox conditions. Scale bar, 200 µm. The experiment was performed in 3 independent XIST iKD clones of H9 hESCs. Out of which one is shown here and the other two are shown in figure S5E (total n=3 independent clones). C. Representative IF of GATA3 (Green) of cells mentioned in 4B. DAPI is shown in blue. Scale bar, 20 µm. The experiment was performed in 3 independent XIST iKD clones of H9 hESCs. Out of which one is shown here and the other two are shown in figure S5F (total n=3 independent clones). D. The proportion of GATA3 positive nuclei in both WT and XIST iKD H9 hESCs at Day 15 of trophoblast differentiation with or without Dox. The number of nuclei counted is indicated above each bar. Each dot represents one of three independent XIST iKD clones (n=3) or WT clones (n=3). Statistical significance was assessed using a t-test. A p-value <0.05 was considered statistically significant. The number of nuclei counted is indicated in supplemental table S1. E. Flow cytometry quantification of the proportion of ENPEP positive cells at day 15 of trophoblast differentiation of XIST iKD hESCs with or without Dox. Statistical significance was tested using a Chi² test, resulting in p<0,0001. Each dot represents one of three independent XIST iKD clones (n=3). F. UMAP of scRNA-seq time course during differentiation of H9 WT and XIST iKD hESCs with or without Dox under trophoblast culture conditions, 13 clusters are shown using different colors. The scRNA-seq data was generated using one of the above 3 clones both for the WT and the XIST iKD hESCs with or without Dox (n=1). G. As in 4F but colored by cell types. Lineage information where Naive = Naive epiblast, Differ Naive = Differentiated naive epiblast, Epi interim 1 = Epiblast intermediate type 1, Epi interim 2 = Epiblast intermediate type 2, Epi interim 3 = Epiblast intermediate type 3, Epi interim 4 = Epiblast intermediate type 4, TSC = Trohpblast stem cells, and EXMC = Extraembryonic mesoderm cells. H. UMAPs from showing the expression of KLF4 and DNMT3L (naive hESC markers), GATA3 and GATA2 (TSC markers), LUM and NID2 (EXMC markers) in time course scRNA-seq data. I. Dot plot showing the expression of marker genes for core pluripotency, naive pluripotency, trophoblast and embryo extraembryonic mesoderm (EXM) in time course scRNA-seq data from this study. J. The proportion of each cell type at different time points during differentiation of naive hESCs into TSCs and EXMCs. Colored by cell types. K. Differential gene expression as detected by scRNA-seq between Day 8 XIST iKD (Top) and WT cells (Bottom) with Dox vs without Dox. Dashed lines indicate -log10 adjusted p-Value <0.05 and log2 fold change < -0.5 or > 0.5. L. GO enrichment analysis of differentially expressed genes as a result of XIST depletion comparing Day 8 XIST iKD cells with and without Dox.

Article Snippet: In summary, starting from day 1 or day 2 after seeding primed hESCs in E8 onto Geltrex, cells were lipofected daily with KLF4 mRNA (Miltenyi, 130-101-115) for 9 days .

Techniques: Knockdown, Microscopy, RNA Sequencing, Flow Cytometry, Clone Assay, Generated, Expressing, Marker, Gene Expression

Journal: Human Reproduction Open

Article Title: Human iPSCs derived from cryopreserved testicular somatic cells enable germline regeneration in childhood cancer survivors

doi: 10.1093/hropen/hoag054

Figure Lengend Snippet: Overview of the experimental layout: focus on primary testicular somatic cell cultures and reprogramming . ( A ) Schematic overview of the experimental procedures. ( B ) Non-genome-integrating, feeder-free reprogramming of human induced pluripotent stem cells (hiPSCs) from primary testicular somatic cells, with the corresponding time required for each step. Left panel: Phase-contrast images of confluent primary testicular somatic cell cultures approximately 1 week after isolation from the cryopreserved tissue; Middle panel: Emerging hiPSC colonies following the mRNA-based reprogramming protocol; Right panel: Expansion of the isolated hiPSC clones NFKI001 at passage 8 (p8) and NFKI002 at p15 from Patients 1 and 2, respectively. Scale bars, 500 μm. Figure (A) was created using BioRender (Alves Lopes, J. P. (2026); https://BioRender.com/gv6k171 ).

Article Snippet: Upon reaching confluency, cells were seeded at 2000 cells/cm 2 in 35 mm dishes on day-3 and transfected twice daily from day 0 to day 4 using the StemMACSTM iPSC mRNA Reprogramming Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-132-990).

Techniques: Isolation, Clone Assay