Journal: Neurochemical Research

Article Title: Nrf2 and STAT3 Pathways in Protecting Against Methylmercury-Induced Oxidative Stress in Hypothalamic Neuronal GT1-7 Cells

doi: 10.1007/s11064-026-04898-1

Figure Lengend Snippet: Inhibition of STAT3 enhances MeHg-induced Nrf2 activity in mouse hypothalamic neuronal GT1-7 cells. A Representative western blot analysis of Nrf2 protein levels in cytoplasmic and nuclear fractions, with densitometry quantification of nuclear Nrf2 protein levels in GT1-7 cells pre-treated with 100 µM AG490 or 30 µM C188-9 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 9 independent cell culture preparations). B qPCR analysis of Nfe2l2 mRNA levels in GT1-7 cells treated with 10 or 100 µM AG490 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations). C qPCR analysis of Keap1 mRNA levels in GT1-7 cells treated with 10 or 100 µM AG490 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations). D qPCR analysis of Slc7a11 mRNA levels in GT1-7 cells treated with 10 or 100 µM AG490 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations). E qPCR analysis of Nqo1 mRNA levels in GT1-7 cells treated with 10 or 100 µM AG490 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations). F qPCR analysis of Nfe2l2 mRNA levels in GT1-7 cells treated with 15 or 30 µM C188-9 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations).) qPCR analysis of Keap1 mRNA levels in GT1-7 cells treated with 15 or 30 µM C188-9 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations). H qPCR analysis of Slc7a11 mRNA levels in GT1-7 cells treated with 15 or 30 µM C188-9 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 6–7 independent cell culture preparations). I qPCR analysis of Nqo1 mRNA levels in GT1-7 cells treated with 15 or 30 µM C188-9 for 1 h, followed by co-exposure to 5 µM MeHg for 6 h ( n = 7 independent cell culture preparations). Data are expressed as mean ± SD. * indicates p < 0.05

Article Snippet: To investigate the role of STAT3 in Nrf2 induction, cells were pre-treated for 1 h with either 10 or 100 μM AG490 (Tyrphostin B42, Santa Cruz Biotechnology, Santa Cruz biotechnology, Inc. cat. no. sc-202046) or with 15 or 30 μM of STAT3 Inhibitor XIII, C188-9 (CalbioChem, cat. no. 573128).

Techniques: Inhibition, Activity Assay, Western Blot, Analysis, Cell Culture

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , Experimental design of the CRISPR–Cas9 screen across the mouse ζ-globin ( Hba-x ) gene promoter. b , Normalization of mVenus + (%) to editing efficiency reveals two potential repressive regions, highlighted in blue and pink. c , Normalization of Hba-x mRNA (%) to editing efficiency in G1E-ER4 cells identifies two potential repressive cis -regulatory elements, targeted by sg33 and sg39/sg40/sg41. Data are presented as mean values ( n = 2 biological replicates). d , Percentage of Hba-x mRNA to total α-like globin in clonal G1E-ER4 cell lines (3 independent single mutant clones, 3 independent replicates of a double-mutant clone). Data are presented as mean ± s.d. Exact P values are shown; statistical significance was assessed using one-way ANOVA with Dunnett correction for multiple comparisons. TSS, transcription start site; ANOVA, analysis of variance.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: CRISPR, Mutagenesis, Clone Assay, Analysis

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a – c , Gating strategy ( a ) for experiment presented in Fig. . Cell viability ( b ) mVenus + (%) and editing efficiency ( c ) in fetal definitive erythroid cells from the CRISPR screen. d , Percentage of Hba-x mRNA to total α-like globin (upper) and sgRNA editing efficiency (lower) in G1E-ER4 cells from the CRISPR screen ( n = 2 biological replicates). Data are presented as mean values. e , Identified repressive elements in the mouse Hba-x gene promoter align to BCL11A and LRF binding motifs from the listed references. f , DNA sequences of the Hba-x gene promoter of G1E-ER4 clonal cell lines. Dashes indicate deletions, and converted bases are shown in red.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: CRISPR, Binding Assay

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , Hematological parameters of homozygous ZPE mice compared to WT mice ( n = 3 male mice aged 13–15 weeks). Data are presented as mean ± s.d. b , α-like globin mRNA expression in fetal liver cells from WT and ZPE homozygous embryos at E13.5 and E14.5 (n = 4 ZPE homozygous E14.5 embryos, n = 3 embryos for other groups). Data are presented as mean ± s.d. Exact P values are shown in the figure; statistical significance was assessed using two-way ANOVA with Bonferroni correction for multiple comparisons. c , Percentage of Hba-x mRNA to total β-like globin in primitive blood from WT and ZPE homozygous E11.5-E12.5 embryos ( n = 4 biological replicates). d , β-like globin mRNA expression in the WT, heterozygous and homozygous -- SEA deletion mouse models ( n = 5, 5 and 3 biological replicates, respectively). Data are presented as mean ± s.d.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: Expressing

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , Sanger sequencing results showing the Hba-x gene promoter sequence in the ZPE mouse model. Deletions of BCL11A and LRF motifs are indicated. b , Percentage of Hba-x mRNA to total β-like globin in E14.5 fetal liver cells and Ter119 + bone marrow cells from WT and ZPE homozygotes ( n = 4 ZPE homozygous E14.5 embryos, n = 3 biological replicates for other groups). Data are presented as mean ± s.d. Bone marrow cells were isolated from male mice aged 13–15 weeks. c , ATAC–seq across the α-globin locus (mm10, chr11: 32,185,000–32,329,000) in fetal definitive erythroblasts from E12.5 WT and BHFS mouse embryos. The Hba-x gene is highlighted in green, the -- SEA deletion is highlighted in blue. The α-globin enhancers (R1–R4, Rm) are indicated. d , α-like globin mRNA expression in the WT, heterozygous and homozygous -- SEA deletion mouse models ( n = 5, 6 and 4 biological replicates, respectively). Data are presented as mean ± s.d. P values are shown; statistical significance was assessed using two-way ANOVA with Dunnett correction for multiple comparisons. e , Percentage of Hba-x mRNA to total β-like globin in peripheral blood from WT, BHFS and BHFS + ZPE E17.5 embryos ( n = 3, 3 and 2 biological replicates, respectively). Data are presented as mean values. f , Isoelectric focusing of extracts from E17.5 peripheral blood of WT, BHFS and BHFS + ZPE embryos. Extracts from E14.5 peripheral blood (lane 1) are used as mouse hemoglobin markers and positive controls for Hb Portland 2 (ζ 2 β 2 ). Experiments were independently repeated thrice with similar results. g , Representative microscopy of WT and BHFS mouse embryos at E15.5–E17.5, white triangles indicate signs of edema. Scale bar = 2 mm. Next to the embryos, representative cytospins of peripheral blood from E17.5 embryos stained using a modified Wright’s procedure. Scale bar = 10 μm. Right, flow cytometry analysis of erythroid enucleation of peripheral blood from E17.5 embryos. Enucleated cells are identified as Hoechst 33342-negative cells. FSC, forward scatter. M, million. Similar results were obtained from at least two independent embryos.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: DNA Sequencing, Sequencing, Isolation, Expressing, Microscopy, Staining, Modification, Flow Cytometry, Analysis

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , Schematic structures of the ζ-globin gene promoter of mouse ( Hba-x , top) and human ( HBZ , bottom). Binding motifs for BCL11A, LRF, GATA1 and NF-Y are indicated. b , Percentage of HBZ mRNA to total α-like globin in WT and mutant HUDEP-2 cell lines ( n = 3 biological replicates for WT, n = 5, 4 and 3 independent clones for distal BCL11A motif mutant, LRF motif mutant and proximal BCL11A motif mutant, respectively) before and after differentiation (day 8). Data are presented as mean ± s.d. Exact P values are shown; statistical significance was assessed using two-way ANOVA with Dunnett correction for multiple comparisons. c , Quantification of HBZ mRNA during erythroid differentiation of CD34 + HSPCs, base-edited cells are in bulk populations ( n = 3 biological replicates). Data are presented as mean ± s.d. d , Percentage of HBZ mRNA to total α-like globin in clonal HUDEP-2 cell lines with mutations at both the distal BCL11A and the LRF binding sites (double mutant, n = 4 independent clones). WT cells are presented as controls (WT, n = 3 biological replicates). Data are presented as mean ± s.d. Exact P value is shown; statistical significance was assessed using an unpaired two-tailed t test. e , Intracellular staining using Alexa Fluor 647-labeled ζ-globin antibody shows that double-mutant HUDEP-2 cell lines produce ζ-globin protein in a pan-cellular manner. f , Isoelectric focusing of extracts from WT (lane 1) and four independent double-mutant HUDEP-2 clones (lanes 2–5) on day 10 of differentiation, showing production of hemoglobins. An FASC control (lane 6), extracts of blood from a transfused BHFS patient (lane 7) and a healthy donor (lane 8) are used as markers. Hemoglobin molecules are indicated.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: Binding Assay, Mutagenesis, Clone Assay, Two Tailed Test, Staining, Labeling, Control

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , ATAC–seq tracks across the human α-globin locus (hg38, chr16: 80,000–210,000) in HUDEP-2 cells on day 8 of differentiation. The ζ-globin ( HBZ ) gene is highlighted in green; the -- SEA deletion is highlighted in blue. b , mRNA quantification of globin genes in WT and BHFS HUDEP-2 cells on day 8 of differentiation ( n = 3 biological replicates). Data are presented as mean ± s.d. Exact P value is shown; statistical significance was assessed using two-way ANOVA with Bonferroni correction for multiple comparisons. c , Percentage of HBZ mRNA to total β-like globin in WT, BHFS ( n = 3 biological replicates) and clonal BHFS HUDEP-2 cell lines with mutations at both the distal BCL11A and the LRF binding sites (BHFS + DM, n = 3 independent clones). Data are presented as mean ± s.d. Exact P values are shown; statistical significance was assessed using one-way ANOVA with Bonferroni correction for multiple comparisons. d , Intracellular staining using Alexa Fluor 647-labeled ζ-globin antibody shows slightly increased ζ-globin protein expression in the BHFS HUDEP-2 cell model, and the promoter double mutation (BHFS + DM) further induces ζ-globin expression.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: Binding Assay, Clone Assay, Staining, Labeling, Expressing, Mutagenesis

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , Mutation of both repressive motifs (Double Mut) increases HBZ mRNA expression in CD34 + HSPC-derived erythroid cells from healthy donors and HbH patients ( n = 3 biological replicates). P values are shown; statistical significance was assessed using two-way ANOVA with Bonferroni correction for multiple comparisons. b , Base-editing quantification using EditR shows representative editing efficiencies at the BCL11A and LRF motifs in adult HSPCs using ABE or CBE. c , Percentage of α-like globin to β-like globin in erythroid cells derived from HbH patients ( n = 3 biological replicates), HBZ mRNA expression is normalized to editing efficiencies. Data are presented as mean ± s.d. d , Mutation of both repressive motifs increases HBZ mRNA in HSPC-derived erythroid cells from a 21-week BHFS fetus (4 independent experiments). Expression of BCL11A isoforms in these cells is unknown. Data are presented as mean ± s.d. Exact P value is shown; statistical significance was assessed using a paired two-tailed t test. e , Base-editing quantification using EditR shows representative editing efficiencies at the BCL11A and LRF motifs in fetal HSPCs. f , Intracellular staining using Alexa Fluor 647-labeled ζ-globin antibody shows increased ζ-globin protein expression in double-mutant erythroid cells (DM, in bulk) derived from HbH and BHFS patients.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: Mutagenesis, Expressing, Derivative Assay, Two Tailed Test, Staining, Labeling

Journal: Nature Genetics

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

doi: 10.1038/s41588-026-02770-0

Figure Lengend Snippet: a , Representative flow cytometry analysis of HSPC-derived erythroid cells from the healthy donor, HbH patient and BHFS fetus, on day 13 of differentiation. b , Representative cytospins of HSPC-derived erythroid cells on day 17 of differentiation, stained using a modified Wright’s procedure, scale bar = 10 μm. c , Mutation of both repressive motifs increases HBZ mRNA in HSPC-derived erythroid cells from a 21-week BHFS fetus (4 independent experiments). Data are presented as mean ± s.d. Exact P values are shown in the figure; statistical significance was assessed using a paired two-tailed t-test.

Article Snippet: Base editor mRNA was produced by in vitro transcription using the HiScibe T7 mRNA (NEB) following the manufacturer’s protocol.

Techniques: Flow Cytometry, Analysis, Derivative Assay, Staining, Modification, Mutagenesis, Two Tailed Test