krab dcas9 mecp2 cassette  (Addgene inc)

Journal: Developmental Neurobiology

Article Title: Vagus Nerve Stimulation Paired With Tones Alters the Auditory Cortex Proteome in a Rat Model of Rett Syndrome

doi: 10.1002/dneu.70032

Figure Lengend Snippet: (A) Auditory cortex protein expression in heterozygous Mecp2 +/ − rats relative to WT controls. Each dot represents a protein identified. Proteins in green are differentially expressed (i.e., p adjusted < 0.05 and/or fold change > 2). Vertical lines represent a log2(two‐fold change), and the horizontal line represents −log( p adjusted < 0.05). (B) The top 20 (10 upregulated and 10 downregulated) differentially expressed proteins were z ‐scored with SEM shown in the error bars.

Article Snippet: Similarly, after VNS‐tone pairing in Mecp2 +/− rats, a significant increase in neural response strength and decrease in neural response latency evoked by tones and speech sounds in VNS+ Mecp2 +/− rats relative to untreated Mecp2 +/ − rats was observed (Adcock et al. ).

Techniques: Expressing

Journal: Developmental Neurobiology

Article Title: Vagus Nerve Stimulation Paired With Tones Alters the Auditory Cortex Proteome in a Rat Model of Rett Syndrome

doi: 10.1002/dneu.70032

Figure Lengend Snippet: Protein‒protein interactions of differentially expressed auditory cortex proteins in heterozygous Mecp2 +/ − rats relative to WT controls. Proteins are identified as nodes, and associations are identified by the connecting lines. The relative confidence in the association is depicted by the width of the line, with dotted lines representing predicted connections between clusters. Primary clusters of interest are identified in color. The protein clusters relating to energy metabolism are shown in red (pentose phosphate and glycolysis shown in dark red, citrate cycle shown in light red), and the clusters relating to vesicular transport are shown in green (Rab GTPases are shown in dark green, SNARE proteins are shown in light green).

Article Snippet: Similarly, after VNS‐tone pairing in Mecp2 +/− rats, a significant increase in neural response strength and decrease in neural response latency evoked by tones and speech sounds in VNS+ Mecp2 +/− rats relative to untreated Mecp2 +/ − rats was observed (Adcock et al. ).

Techniques:

Journal: Developmental Neurobiology

Article Title: Vagus Nerve Stimulation Paired With Tones Alters the Auditory Cortex Proteome in a Rat Model of Rett Syndrome

doi: 10.1002/dneu.70032

Figure Lengend Snippet: (A) Auditory cortex protein expression in VNS+ Mecp2 +/ − rats relative to untreated Mecp2 +/ − rats. Each dot represents a protein identified. Proteins in green are differentially expressed (i.e., p adjusted < 0.05 and/or fold change > 2). Vertical lines represent a log2(two‐fold change), and the horizontal line represents −log( p adjusted < 0.05). (B) The top 20 (10 upregulated and 10 downregulated) differentially expressed proteins were z ‐scored with SEM shown in the error bars.

Article Snippet: Similarly, after VNS‐tone pairing in Mecp2 +/− rats, a significant increase in neural response strength and decrease in neural response latency evoked by tones and speech sounds in VNS+ Mecp2 +/− rats relative to untreated Mecp2 +/ − rats was observed (Adcock et al. ).

Techniques: Expressing

Journal: Developmental Neurobiology

Article Title: Vagus Nerve Stimulation Paired With Tones Alters the Auditory Cortex Proteome in a Rat Model of Rett Syndrome

doi: 10.1002/dneu.70032

Figure Lengend Snippet: Protein‐to‐protein interactions of differentially expressed auditory cortex proteins in VNS+ Mecp2 +/ − rats relative to untreated Mecp2 +/ − rats. Proteins are identified as nodes, and associations are identified by the connecting lines. The relative confidence in the association is depicted by the width of the line, with dotted lines representing predicted connections between clusters. Primary clusters of interest are identified in color. The protein cluster related to aerobic respiration is shown in red, the protein cluster related to synaptic regulation is shown in light blue, the protein cluster related to glycolysis is shown in yellow, the protein cluster related to myelination is shown in dark blue, and the protein cluster related to synaptic vesicle endocytosis is shown in green.

Article Snippet: Similarly, after VNS‐tone pairing in Mecp2 +/− rats, a significant increase in neural response strength and decrease in neural response latency evoked by tones and speech sounds in VNS+ Mecp2 +/− rats relative to untreated Mecp2 +/ − rats was observed (Adcock et al. ).

Techniques:

Journal: Developmental Neurobiology

Article Title: Vagus Nerve Stimulation Paired With Tones Alters the Auditory Cortex Proteome in a Rat Model of Rett Syndrome

doi: 10.1002/dneu.70032

Figure Lengend Snippet: (A) Auditory cortex protein expression in VNS+ Mecp2 +/ − rats relative to WT controls. Each dot represents a protein identified. Proteins in green are differentially expressed (i.e., p adjusted < 0.05 and/or fold change > 2). Vertical lines represent a log2(two‐fold change), and the horizontal line represents −log( p adjusted < 0.05). (B) The top 20 (10 upregulated and 10 downregulated) differentially expressed proteins were z ‐scored with SEM shown in the error bars.

Article Snippet: Similarly, after VNS‐tone pairing in Mecp2 +/− rats, a significant increase in neural response strength and decrease in neural response latency evoked by tones and speech sounds in VNS+ Mecp2 +/− rats relative to untreated Mecp2 +/ − rats was observed (Adcock et al. ).

Techniques: Expressing

Journal: Developmental Neurobiology

Article Title: Vagus Nerve Stimulation Paired With Tones Alters the Auditory Cortex Proteome in a Rat Model of Rett Syndrome

doi: 10.1002/dneu.70032

Figure Lengend Snippet: Protein‐to‐protein interactions of differentially expressed auditory cortex proteins in VNS+ Mecp2 +/ − rats relative to WT controls. Proteins are identified as nodes, and associations are identified by the connecting lines. The relative confidence in the association is depicted by the width of the line, with dotted lines representing predicted connections between clusters. Primary clusters of interest are identified in color. The protein cluster relating to the TCA cycle is shown in red, the protein cluster relating to cytoskeleton proteins is shown in yellow, the protein cluster relating to mitochondrial regulation is shown in blue, the protein cluster relating to myelin formation is shown in orange, and the endocytosis protein cluster is shown in green.

Article Snippet: Similarly, after VNS‐tone pairing in Mecp2 +/− rats, a significant increase in neural response strength and decrease in neural response latency evoked by tones and speech sounds in VNS+ Mecp2 +/− rats relative to untreated Mecp2 +/ − rats was observed (Adcock et al. ).

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2

doi: 10.1016/j.omtn.2026.102844

Figure Lengend Snippet: miR-422a directly targets MECP2 (A) Schematic diagram showing the predicted miR-422a target sites within the MECP2 3′UTR. The predicted target seed sites and their corresponding mutated sequences are highlighted in red. (B) HEK-293T cells were cotransfected with MECP2 3′UTR luciferase reporter vector (wild-type or mutant versions), pRL-TK, and miR-422a mimic or negative control (NC) for 24 h. Cells were then harvested for luciferase analysis. Relative luciferase activity is shown. (C) Unstimulated or stimulated primary CD4+ T cells were transfected with miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 mRNA levels by RT-qPCR. (D) Stimulated primary CD4+ T cells were transfected with the indicated concentrations of miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 protein levels by western blot. β-actin was used as the endogenous control. (E) Relative quantification of MECP2 protein from the western blot in (D). (F) MECP2 protein expression was analyzed by western blot following the establishment of stable cell lines. (G) MECP2 KO-Jurkat or scramble-Jurkat cells were infected with HIV-1 NL4-3 . The percentage of Gag-positive cells was detected by flow cytometry at the indicated time point. (H and I) Stimulated primary CD4+ T cells were nucleofected with Cas9-RNP targeting MECP2 for 24 h, then infected with HIV-1 NL4-3 for 6 days. Cells and supernatants were collected for Gag detection and p24 measurements by flow cytometry (H) and ELISA (I), respectively. (J) MECP2 KO-Jurkat or scramble-Jurkat cells were transfected with miR-422a mimic or its NC for 24 h, then infected with HIV-1 NL4-3 (or left uninfected). Six days after infection, cells were collected, fixed, and permeabilized, then stained with RD-fluorescent Gag antibody to detect Gag expression by flow cytometry. The percentage of Gag-positive cells was determined by flow cytometry. Each dot represents data from one donor. Data are representative of the results of three independent experiments ( n = 3 biologically independent samples, mean ± SEM). Statistical significance was analyzed by unpaired or paired student t tests. p ≤ 0.05 [∗], p ≤ 0.01 [∗∗], p ≤ 0.001 [∗∗∗], p ≤ 0.0001 [∗∗∗∗].

Article Snippet: To confirm that MECP2 is a target of miR-422a, a fragment of the MECP2 3′UTR containing the predicted target site was amplified and cloned downstream of the firefly luciferase gene in the pGL-3-control vector (Addgene, #212937).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Negative Control, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Expressing, Stable Transfection, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2

doi: 10.1016/j.omtn.2026.102844

Figure Lengend Snippet: MECP2 counteracts IFNα activity (A) Wild-type Jurkat cells (Jurkat), MECP2 KO-Jurkat cells, or scramble-Jurkat cells were treated with or without IFNα for 6 h. Cells were collected to detect MECP2, phosphorylation of STAT3, and STAT3 protein levels by western blot. GAPDH was used as the endogenous control. (B) Relative quantification of MECP2, phosphorylated STAT3, and total STAT3 protein from western blot (A). (C) MECP2 KO-Jurkat or scramble-Jurkat cells were treated with or without IFNα for 72 h. Cells were collected to detect IFIT1 expression by RT-qPCR. (D) MECP2 KO-Jurkat or scramble-Jurkat cells were treated with or without IFNα for 24 h, then infected with HIV-1 NL4-3 and treated again with IFNα at the same time. The percentage of Gag-positive cells was determined by flow cytometry at each time point post HIV-1 NL4-3 infection. (E) Stimulated primary CD4+ T cells were nucleofected with Cas9 RNP targeting MECP2 for 24 h, then treated with IFNα for 72 h. Cells were collected to detect ISG15 expression by RT-qPCR. (F) Stimulated primary CD4+ T cells were nucleofected with Cas9 RNP targeting MECP2 for 24 h, then infected with HIV-1 NL4-3 and treated with IFNα at the same time. The percentage of Gag-positive cells was determined by flow cytometry after 6 days of HIV-1 infection. Each dot represents data from one donor. Data are representative of the results of three independent experiments ( n = 3 biologically independent samples, mean ± SEM). Statistical significance was analyzed by unpaired or paired student t tests. p ≤ 0.05 [∗], p ≤ 0.01 [∗∗], p ≤ 0.001 [∗∗∗], p ≤ 0.0001 [∗∗∗∗].

Article Snippet: To confirm that MECP2 is a target of miR-422a, a fragment of the MECP2 3′UTR containing the predicted target site was amplified and cloned downstream of the firefly luciferase gene in the pGL-3-control vector (Addgene, #212937).

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Infection, Flow Cytometry

Journal: Science translational medicine

Article Title: Modulating alternative splicing of MECP2 is a potential therapeutic strategy for Rett syndrome

doi: 10.1126/scitranslmed.adq4529

Figure Lengend Snippet: (A) Schematic of MECP2 exons alternatively spliced into e1 and e2 at pre-mRNA level. Purple denotes e1 and orange denotes e2 . Translational start sites of e1 and e2 are indicated by a black arrow on mature mRNA. E1 and E2 proteins have distinct N-termini but identical functional domains. (B) e1 and e2 absolute mRNA concentrations (left), Western blot showing E1 and E2 protein bands (center), E1 and E2 protein quantifications and protein/mRNA ratios (right) in post-mortem human prefrontal cortex tissues (n= 3 males (triangles) and 2 females (circles)), statistical analyses done using paired t-tests (*p<0.05). Data are presented as mean±sem. Error bars represent sem. Individual datapoints representing biological replicates. (C) Schematic representation of how isoform switching in MECP2 by exon 2 deletion abolishes e2 mRNA, upregulates e1 mRNA, thereby upregulating E1 protein.

Article Snippet: We used the anti-MeCP2 antibody that detects both isoformsx( ) (Cell Signaling Technology #3456, 1:1000).

Techniques: Functional Assay, Western Blot

Journal: Science translational medicine

Article Title: Modulating alternative splicing of MECP2 is a potential therapeutic strategy for Rett syndrome

doi: 10.1126/scitranslmed.adq4529

Figure Lengend Snippet: (A) e1 and e2 absolute mRNA concentrations (left), Western blot showing MeCP2 protein bands (center) and quantification (right) in cortices of wild-type–black (n=4) and E2KO–green (n=6) mice (B) Elevated plus maze (EPM) analysis: time in closed arms (left) and number of entries into open arms (right) (C) Open field assay (OFA): total distance travelled (left) and normalized distance (distance in center/total distance) (right) (D) Rotarod assay: latency to fall (in seconds) across 4 test days, 4 trials per day (E) Percent freezing in the conditioned fear paradigm in response to cue (left) and context (right). Behavioral assays were performed on 12 wild-type and 19 E2KO male mice. Statistical analyses: panels A,B,C, E: unpaired t-test and panel D: two-way ANOVA with multiple comparisons (ns: p>0.05, * p<0.05, ** p<0.001). Data are presented as mean±sem. Error bars represent sem. Individual datapoints represent biological replicates.

Article Snippet: We used the anti-MeCP2 antibody that detects both isoformsx( ) (Cell Signaling Technology #3456, 1:1000).

Techniques: Western Blot

Journal: Science translational medicine

Article Title: Modulating alternative splicing of MECP2 is a potential therapeutic strategy for Rett syndrome

doi: 10.1126/scitranslmed.adq4529

Figure Lengend Snippet: (A) Schematic of G118E, control and G118E-E2KO NGN2-iNeurons derivation from G118E patient fibroblasts. G118E fibroblasts were reprogrammed into G118E iPSCs (in red), which were then edited by CRISPR/Cas9 editing to correct the G118E mutation to WT to generate isogenic control iPSCs (in grey). Exon 2 was deleted by CRISPR/Cas9 editing in G118E iPSCs to generate G118E-E2KO iPSCs (in blue). These three sets of iPSCs were sequentially infected with lentivirus containing doxycycline-inducible rtTA and NGN2 constructs and exposed to doxycycline to overexpress NGN2 and induce neuronal differentiation to generate NGN2-iNs. Figure created with Biorender.com . (B) Representative immunofluorescence images of NGN2-iNeurons (Control, G118E and G118E-E2KO) at 8 weeks of differentiation stained for the neuronal maturation marker MAP2 (red) and DAPI nuclear stain (in blue) (Scale bar: 50μm) (C) e1 (purple) and e2 (orange) mRNA proportions measured by qRT-PCR in control, G118E and G118E-E2KO NGN2-iNeurons (D) Left – Western blot showing MeCP2 and GAPDH (internal control) proteins in control, G118E and G118E-E2KO NGN2-iNs (N=4 technical replicates per genotype), right - MeCP2 quantification relative to GAPDH. Statistical analysis in panel D was performed by two-way ANOVA with multiple comparisons (ns: p>0.05, * p<0.05, ** p<0.01). Data are presented as mean±sem. Error bars represent sem. Individual datapoints represent replicate wells.

Article Snippet: We used the anti-MeCP2 antibody that detects both isoformsx( ) (Cell Signaling Technology #3456, 1:1000).

Techniques: Control, CRISPR, Mutagenesis, Infection, Construct, Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Western Blot

Journal: Science translational medicine

Article Title: Modulating alternative splicing of MECP2 is a potential therapeutic strategy for Rett syndrome

doi: 10.1126/scitranslmed.adq4529

Figure Lengend Snippet: (A) Schematic of Morpholino treatment paradigm in HEK293T cells (created with Biorender.com ) (B) Left - Western blot showing bands for MeCP2 and GAPDH (internal control) in HEK293T cells treated with Control or E2Skip Morpholino (N=3 each), right - quantification of MeCP2 normalized to GAPDH in these cells (C) Schematic of Morpholino treatment paradigm in P0 FVB wild-type mouse cortices (created with Biorender.com ) (D) Left - Western blot bands showing MeCP2 and GAPDH in wild-type FVB male mice cortices injected at P0 with Control Mo or E2Skip Mo (n=3 each) and harvested 2 weeks post-injection, right - quantification of MeCP2 normalized to GAPDH in these tissues. Statistical analyses were done using unpaired t-tests (*p<0.05). Data are presented as mean±sem. Error bars represent sem. Individual datapoints represent biological replicates.

Article Snippet: We used the anti-MeCP2 antibody that detects both isoformsx( ) (Cell Signaling Technology #3456, 1:1000).

Techniques: Western Blot, Control, Injection

Journal: bioRxiv

Article Title: mPFC pyramidal neuron synchrony during social competition to form social rankings is disrupted in male Mecp2 knockout mice

doi: 10.64898/2026.03.02.709145

Figure Lengend Snippet: (A) Social hierarchy between 3 age-matched male mice of the same genotype was determined using the tube test (3 days of training alone and 6 days of round-robin style tournaments between male mice of the same genotype)5 . (B, D) Cumulative percentages of trial outcomes classifying the 3 male WT mice (B) and male Mecp2 KO mice (D) in each cage into dominant (DOM), intermediate (INT), and subordinate (SUB), based in their percentages of wins, loses, and ties after 6 days of tournament. (C, E) EloRating score in each tournament day for male WT mice (C) and male Mecp2 KO mice (E); EloRating scores start as 1,000. Analyzing performance during the tube test tournament, we found that male Mecp2 KO mice have atypical behaviors during the tube test to solve this social conflict: they spent more time inside the tube (F), engaged in fewer body contacts inside the tube (G), initiated fewer pushes (H), and fewer push-back as response to pushing (I), engaged in fewer resistances (J), no differences during the retreat time (K), and spent less time chasing the opponent (L). Hierarchy score was calculated as a composed measurement of dominant behaviors, similar to previously reported [ , ]. We calculated the z-scores from total number of wins during pairwise tournament, pushing , push-back , resistance and chasing behavior. Once each z-score were calculated were combined in the hierarchy score. A high punctuation in this hierarchy score means that mice showed more dominant behaviors after tournament, punctuations close to zero or negative mean submissive or lack of dominance. Data in F-M were analyzed with a mixed factorial ANOVA model. * p < 0.05

Article Snippet: Female mice from the Jaenisch strain with a deletion of exon 3 of the Mecp2 gene (Mecp2tm1.1Jae) [ ])were obtained from the Mutant Mouse Resource & Research Center , and a colony established at the University of Alabama at Birmingham (UAB) by crossing them with male WT C57/BL6 mice.

Techniques:

Journal: bioRxiv

Article Title: mPFC pyramidal neuron synchrony during social competition to form social rankings is disrupted in male Mecp2 knockout mice

doi: 10.64898/2026.03.02.709145

Figure Lengend Snippet: (A) Diagram of modified surgery to implant pre-coated-GRIN lens in the mPFC of WT and Mecp2 KO mice, followed by tube test tournament to determinate their social ranking, and the warm spot test 24 hours after. (B) Example of pre-coated-GRIN lens placement and jGCaMP8m-expressing excitatory neurons ( CaMKIIa promoter). (C) Representative FOV of maximal projection in WT mouse (C top) after motion correction and CNMFe processing. (D top) Representative jGCaMP8m traces (% dF/F) during the training in the warm spot test, neurons were randomly selected in each genotype. (C bottom) Representative FOV of maximal projection of Mecp2 KO mice. (D bottom) Representative jGCaMP8m traces (% dF/F) of Mecp2 KO mice. (E - H) Cumulative percentages of trial outcomes classifying the 3 male WT mice (E) and male Mecp2 KO mice (G) in each cage into dominant (DOM), intermediate (INT), and subordinate (SUB). (F, H) EloRating score in each tournament day for male WT mice (F) and male Mecp2 KO mice (H); (I, J) Hierarchy scores calculated during the tube test (I), and after the warm spot test (J) showed that WT mice exhibited more dominant behaviors after tournament and during the warm spot test. (K, L) The frequency of jGCaMP8m events during habituation (baseline) and the entire warm spot test was significantly lower in Mecp2 KO mice than in WT mice. (M) The amplitude of jGCaMP8m events was significantly lower in Mecp2 KO mice during both habituation and the warm spot test. Behavioral movies were annotated and synchronized with jGCaMP8m movies using BENTO software . To detect neurons responsive to each behavior, each signal above or below two standard deviations from the z-score normalized using a baseline of 3 seconds prior to the onset of each behavior was considered as positively or negatively responsive, respectively. The average trace was compared with the same trace permuted, with the green bar below traces denoting significance p-values calculated by false discovery rates. False Discovery Rates (FDR) compared point-to-point t-tests and corrected for multiple comparisons using significant values satisfying P(k) < 0.05 * k/2000. (O, P) Average dF/F z-scores during nose-to-rear interaction showing average of traces classified as positive responsive in WT mice (O) and in Mecp2 KO (P). (O bottom) Heatmap of dF/F z-scores during nose-to-rear exploration in WT mice, (P bottom) in Mecp2 KO mice. (Q, R) average dF/F z-scores during nose-to-rear interaction showing average of traces classified as negative responsive in WT mice (Q top) and in Mecp2 KO (R). (S, T) Positive responsive traces in WT mice (S top) and in Mecp2 KO mice (T top) during pushing behavior. Negative responsive (U and V). (W -Y). Percentage of positive, negative and non-responsive neurons during different social behaviors was similar in the mPFC of Mecp2 KO (n = 9/21 mice) and WT mice (n = 13/21 mice). Scale bar in panel C correspond to 100 μm. * p < 0.05.

Article Snippet: Female mice from the Jaenisch strain with a deletion of exon 3 of the Mecp2 gene (Mecp2tm1.1Jae) [ ])were obtained from the Mutant Mouse Resource & Research Center , and a colony established at the University of Alabama at Birmingham (UAB) by crossing them with male WT C57/BL6 mice.

Techniques: Modification, Spot Test, Expressing, Software

Journal: bioRxiv

Article Title: mPFC pyramidal neuron synchrony during social competition to form social rankings is disrupted in male Mecp2 knockout mice

doi: 10.64898/2026.03.02.709145

Figure Lengend Snippet: (A) Diagram of surgery to implant pre-coated GRIN lens in the mPFC of WT and Mecp2 KO mice, followed by posterior tube test tournament to determine the social ranking. One hour after TT tournament mice were imaging (day 2 to day 6 of TT tournament) while they were exposed to the urine odor of cagemates while they were head-fixed mice. Each mouse was exposed to two cagemates’ urine odors and control odor 10 times per session. Each odor was delivered for 2s followed by 18 s without odor in a randomized trial each day. (B) Representative random calcium traces with the overlaid timing of odor presentations. (C) Representative similarity results across day 2 to day 6 of imaging of the same WT mouse. (D) Representative results of similarity in a Mecp2 KO mouse. (E) Number of vectors across every day. (F) Number of neurons that participate in ensembles every day. (G) Similarity within ensembles. (H) The mean of spikes per second was inferred from the binarization of the raster plot. (I) Correlation of ensembles during each odor exposition.

Article Snippet: Female mice from the Jaenisch strain with a deletion of exon 3 of the Mecp2 gene (Mecp2tm1.1Jae) [ ])were obtained from the Mutant Mouse Resource & Research Center , and a colony established at the University of Alabama at Birmingham (UAB) by crossing them with male WT C57/BL6 mice.

Techniques: Imaging, Control

Journal: bioRxiv

Article Title: mPFC pyramidal neuron synchrony during social competition to form social rankings is disrupted in male Mecp2 knockout mice

doi: 10.64898/2026.03.02.709145

Figure Lengend Snippet: (A) Diagram of CAV2-Cre and DREADD bilateral injections followed by posterior tube test tournament and 24 hours after the ‘Warm Spot’. (B) Representative image of injection sites showing CAV2-Cre in mPFC and DREADDs expression in vHIP neurons. Scale bar 200 µm large, 100 µm inset (top), and 500 µm large, 200 µm inset (bottom). (C, I) Cumulative percentages of trial outcomes classifying the 3 male WT-mCherry mice (C) and male Mecp2 KO-mCherry mice (I) in each cage into dominant (DOM), intermediate (INT), and subordinate (SUB). (D, J) EloRating score in each tournament day for male WT-mCherry mice (D) and male Mecp2 KO-mCherry mice (J); (E, K) Cumulative percentages of trial outcomes classifying the 3 male WT-hM3Dq mice (E) and male Mecp2 KO-hM4Di mice (K). (F, L) EloRating score in each tournament day for male WT-hM3Dq mice (F) and male Mecp2 KO-hM4Di mice (L); (G, H) Hierarchy scores calculated during the tube test (G), and after the Warm Spot test (H) comparing WT-mCherry and WT-hM3Dq showed that WT-mCherry mice exhibited more dominant behaviors after the tournament. (M, N) Hierarchy scores calculated during the tube test (M), and after the Warm Spot test (N) comparing Mecp2 KO-mCherry and Mecp2 KO-hM4Di showed that Mecp2 KO-hM4Di mice exhibited more dominant behaviors after the tournament. (O) Hierarchy scores during the tube test for all four groups. (P) Hierarchy scores during the ‘warm spot’ for all four groups. * p < 0.05 with posterior post hoc comparison using Tukeýs HSD.

Article Snippet: Female mice from the Jaenisch strain with a deletion of exon 3 of the Mecp2 gene (Mecp2tm1.1Jae) [ ])were obtained from the Mutant Mouse Resource & Research Center , and a colony established at the University of Alabama at Birmingham (UAB) by crossing them with male WT C57/BL6 mice.

Techniques: Injection, Expressing, Spot Test, Comparison

Journal: bioRxiv

Article Title: mPFC pyramidal neuron synchrony during social competition to form social rankings is disrupted in male Mecp2 knockout mice

doi: 10.64898/2026.03.02.709145

Figure Lengend Snippet: (A) To confirm whether social ranking is used to solve social conflicts, the warm spot test was performed during 10 min, previously mice were individually trained in the same warm spot box to sure that each one know where the warm spot was located (B), 48 h posterior to the last day of the tube test tournament. (D, E) In contrast to male WT mice where the DOM mouse stayed the longest on the warm spot (D) and for more instances in each trial (E), male Mecp2 KO mice spent similar amounts of time on the warm spot and for fewer instances, irrespective of their social rank (previously determined in the tube test). (F) The number of winning trials in tube test shows a similar positive correlation with the time spent on the warm spot but without differences between groups. (G - I) During social interactions in the warm spot test Mecp2 mice showed fewer pushing (G), fewer nose-to-nose (H), and fewer nose-to-body interactions (I). (J) Equivalent to the hierarchy score during the tube test, the composed measurement of dominant behaviors during the warm spot test was calculated with z-score from frequency and duration on the warm spot, frequency of pushing , frequency of nose-to-nose , and frequency of nose-to-body . Panels D, E, G, H, I and J show a main effect of group, but not of social rank except to duration on warm spot. * p < 0.05.

Article Snippet: Female mice from the Jaenisch strain with a deletion of exon 3 of the Mecp2 gene (Mecp2tm1.1Jae) [ ])were obtained from the Mutant Mouse Resource & Research Center , and a colony established at the University of Alabama at Birmingham (UAB) by crossing them with male WT C57/BL6 mice.

Techniques: Spot Test

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Morphological relationship between CDX2 and MeCP2 expressions in non-neoplastic duodenal and gastric mucosa. ( a ) Hematoxylin and eosin (HE) staining shows the distinction between the basal glands (asterisk) and the luminal epithelium. ( b ) CDX2 immunostaining highlights nuclear positivity in the luminal epithelium with differentiated morphology. ( c ) MeCP2 immunostaining demonstrates nuclear positivity in the basal glands, showing a reciprocal expression pattern compared with CDX2. ( d ) Higher-magnification views (insets in b and c ) further emphasize this inverse relationship. Stromal cells are largely positive for MeCP2. Bars = 100 μm.

Article Snippet: Primary antibodies against MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) and CDX2 (1:50, DAKO, cat. no. M3636, mouse monoclonal, Glostrup, Denmark) were applied, followed by Alexa Fluor 568-conjugated anti-rabbit (1:2000, Thermo Fisher Scientific, cat. no. A-11011, Waltham, MA, USA) and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:2000, Thermo Fisher Scientific, cat. no. A10680, Waltham, MA, USA).

Techniques: Staining, Immunostaining, Expressing

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: CDX2 and MeCP2 expression in a pancreatobiliary type ampullary carcinoma with morphological heterogeneity. ( a ) Overview of a pancreatobiliary-type carcinoma with focal CDX2 positivity (Bar = 1 mm). ( b ) Well-differentiated tubular adenocarcinoma forming small glandular lumina. ( f ) Well-differentiated adenocarcinoma showing wide glandular lumina with papillary morphology. ( j ) Poorly differentiated adenocarcinoma. ( c , g , k ) CK7 immunostaining confirms preservation of the pancreatobiliary phenotype throughout the tumor. ( d , l ) CDX2 is negative in most regions, whereas ( h ) focal nuclear CDX2 positivity is observed in a subset of CK7-positive cells. ( e ) MeCP2 is diffusely expressed in carcinoma cells, but ( i ) its expression is lost in areas corresponding to CDX2 positivity. Bars = 1 mm ( a ); 100 μm ( b – m ). This case was subjected to Visium analysis .

Article Snippet: Primary antibodies against MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) and CDX2 (1:50, DAKO, cat. no. M3636, mouse monoclonal, Glostrup, Denmark) were applied, followed by Alexa Fluor 568-conjugated anti-rabbit (1:2000, Thermo Fisher Scientific, cat. no. A-11011, Waltham, MA, USA) and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:2000, Thermo Fisher Scientific, cat. no. A10680, Waltham, MA, USA).

Techniques: Expressing, Immunostaining, Preserving

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Visium-based transcriptomic clustering and spatial distribution of MeCP2-associated genes. ( a ) Visium spatial transcriptomic analysis identified five transcriptionally defined spatial clusters using unsupervised principal component analysis followed by graph-based Louvain clustering (Seurat v5.1.0). After clustering, the biological identities of the clusters were interpreted based on histology, with MeCP2 immunohistochemistry overlaid to assist interpretation. Cluster 1 corresponds to non-neoplastic pancreatic parenchyma; Cluster 2 to smooth muscle/mesenchyme; Cluster 3 to well-differentiated carcinoma with MeCP2 nuclear expression; Cluster 4 to well-differentiated carcinoma lacking MeCP2 expression; and Cluster 5 to poorly differentiated carcinoma. Genes consistently enriched in MeCP2-negative regions across two independent analyses are summarized in . ( b – e ) Pseudo– in situ hybridization (spatial feature plots) showing transcript distribution of ( b ) MeCP2, ( c ) TGFB1, ( d ) CDK6, and ( e ) VCAN. CDK6, and VCAN are predominantly expressed in MeCP2-negative clusters (4 and 5). Notably, VCAN-high stromal regions spatially correspond to MeCP2-positive CAFs, supporting a MeCP2-driven stromal remodeling program.

Article Snippet: Primary antibodies against MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) and CDX2 (1:50, DAKO, cat. no. M3636, mouse monoclonal, Glostrup, Denmark) were applied, followed by Alexa Fluor 568-conjugated anti-rabbit (1:2000, Thermo Fisher Scientific, cat. no. A-11011, Waltham, MA, USA) and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:2000, Thermo Fisher Scientific, cat. no. A10680, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing, In Situ Hybridization

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Immunohistochemical localization of VCAN in ampullary carcinoma. ( a , b ) VCAN immunostaining demonstrates epithelial and stromal expression in poorly differentiated adenocarcinoma. ( b , d ) MeCP2 immunostaining shows nuclear positivity in CAFs (red arrows) adjacent to carcinoma cells, whereas carcinoma cells are largely negative (green arrows). These findings indicate that VCAN-positive stromal fibroblasts correspond to MeCP2-positive CAFs. Bars = 20 μm.

Article Snippet: Primary antibodies against MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) and CDX2 (1:50, DAKO, cat. no. M3636, mouse monoclonal, Glostrup, Denmark) were applied, followed by Alexa Fluor 568-conjugated anti-rabbit (1:2000, Thermo Fisher Scientific, cat. no. A-11011, Waltham, MA, USA) and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:2000, Thermo Fisher Scientific, cat. no. A10680, Waltham, MA, USA).

Techniques: Immunohistochemical staining, Immunostaining, Expressing

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: MeCP2 binding to 5hmC-enriched CpG sites in the VCAN promoter. ( a ) Electrophoretic mobility shift assay (EMSA) using two segments (EMSA #1 and EMSA #2) of the 5hmC-enriched region. No significant protein-DNA binding was detected in the absence of 5hmC. ( b ) In the presence of 5hmC, distinct DNA-protein complexes were observed at both EMSA #1 and #2 (arrow). The addition of an anti-MeCP2 antibody induced a block-shift phenomenon (*), confirming specific MeCP2 binding to 5hmC-containing sequences.

Article Snippet: Primary antibodies against MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) and CDX2 (1:50, DAKO, cat. no. M3636, mouse monoclonal, Glostrup, Denmark) were applied, followed by Alexa Fluor 568-conjugated anti-rabbit (1:2000, Thermo Fisher Scientific, cat. no. A-11011, Waltham, MA, USA) and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:2000, Thermo Fisher Scientific, cat. no. A10680, Waltham, MA, USA).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Blocking Assay

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Interaction between MeCP2 and CREB demonstrated by immunoprecipitation. Whole-cell lysates were immunoprecipitated with an anti-MeCP2 antibody, followed by western blotting with an anti-CREB antibody. A distinct band corresponding to CREB was detected at 43 kDa in the MeCP2 immunoprecipitates. No significant bands were observed in the input control (Input) or in immunoprecipitates with a nonspecific rabbit IgG (nR-IgG).

Article Snippet: Primary antibodies against MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) and CDX2 (1:50, DAKO, cat. no. M3636, mouse monoclonal, Glostrup, Denmark) were applied, followed by Alexa Fluor 568-conjugated anti-rabbit (1:2000, Thermo Fisher Scientific, cat. no. A-11011, Waltham, MA, USA) and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:2000, Thermo Fisher Scientific, cat. no. A10680, Waltham, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Morphological relationship between CDX2 and MeCP2 expressions in non-neoplastic duodenal and gastric mucosa. ( a ) Hematoxylin and eosin (HE) staining shows the distinction between the basal glands (asterisk) and the luminal epithelium. ( b ) CDX2 immunostaining highlights nuclear positivity in the luminal epithelium with differentiated morphology. ( c ) MeCP2 immunostaining demonstrates nuclear positivity in the basal glands, showing a reciprocal expression pattern compared with CDX2. ( d ) Higher-magnification views (insets in b and c ) further emphasize this inverse relationship. Stromal cells are largely positive for MeCP2. Bars = 100 μm.

Article Snippet: Primary antibodies against CREB (1:1000, Cell Signaling Technology, cat. no. 9192, rabbit polyclonal, Danvers, MA, USA) and MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) were used.

Techniques: Staining, Immunostaining, Expressing

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: CDX2 and MeCP2 expression in a pancreatobiliary type ampullary carcinoma with morphological heterogeneity. ( a ) Overview of a pancreatobiliary-type carcinoma with focal CDX2 positivity (Bar = 1 mm). ( b ) Well-differentiated tubular adenocarcinoma forming small glandular lumina. ( f ) Well-differentiated adenocarcinoma showing wide glandular lumina with papillary morphology. ( j ) Poorly differentiated adenocarcinoma. ( c , g , k ) CK7 immunostaining confirms preservation of the pancreatobiliary phenotype throughout the tumor. ( d , l ) CDX2 is negative in most regions, whereas ( h ) focal nuclear CDX2 positivity is observed in a subset of CK7-positive cells. ( e ) MeCP2 is diffusely expressed in carcinoma cells, but ( i ) its expression is lost in areas corresponding to CDX2 positivity. Bars = 1 mm ( a ); 100 μm ( b – m ). This case was subjected to Visium analysis .

Article Snippet: Primary antibodies against CREB (1:1000, Cell Signaling Technology, cat. no. 9192, rabbit polyclonal, Danvers, MA, USA) and MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) were used.

Techniques: Expressing, Immunostaining, Preserving

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Visium-based transcriptomic clustering and spatial distribution of MeCP2-associated genes. ( a ) Visium spatial transcriptomic analysis identified five transcriptionally defined spatial clusters using unsupervised principal component analysis followed by graph-based Louvain clustering (Seurat v5.1.0). After clustering, the biological identities of the clusters were interpreted based on histology, with MeCP2 immunohistochemistry overlaid to assist interpretation. Cluster 1 corresponds to non-neoplastic pancreatic parenchyma; Cluster 2 to smooth muscle/mesenchyme; Cluster 3 to well-differentiated carcinoma with MeCP2 nuclear expression; Cluster 4 to well-differentiated carcinoma lacking MeCP2 expression; and Cluster 5 to poorly differentiated carcinoma. Genes consistently enriched in MeCP2-negative regions across two independent analyses are summarized in . ( b – e ) Pseudo– in situ hybridization (spatial feature plots) showing transcript distribution of ( b ) MeCP2, ( c ) TGFB1, ( d ) CDK6, and ( e ) VCAN. CDK6, and VCAN are predominantly expressed in MeCP2-negative clusters (4 and 5). Notably, VCAN-high stromal regions spatially correspond to MeCP2-positive CAFs, supporting a MeCP2-driven stromal remodeling program.

Article Snippet: Primary antibodies against CREB (1:1000, Cell Signaling Technology, cat. no. 9192, rabbit polyclonal, Danvers, MA, USA) and MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) were used.

Techniques: Immunohistochemistry, Expressing, In Situ Hybridization

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Immunohistochemical localization of VCAN in ampullary carcinoma. ( a , b ) VCAN immunostaining demonstrates epithelial and stromal expression in poorly differentiated adenocarcinoma. ( b , d ) MeCP2 immunostaining shows nuclear positivity in CAFs (red arrows) adjacent to carcinoma cells, whereas carcinoma cells are largely negative (green arrows). These findings indicate that VCAN-positive stromal fibroblasts correspond to MeCP2-positive CAFs. Bars = 20 μm.

Article Snippet: Primary antibodies against CREB (1:1000, Cell Signaling Technology, cat. no. 9192, rabbit polyclonal, Danvers, MA, USA) and MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) were used.

Techniques: Immunohistochemical staining, Immunostaining, Expressing

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: MeCP2 binding to 5hmC-enriched CpG sites in the VCAN promoter. ( a ) Electrophoretic mobility shift assay (EMSA) using two segments (EMSA #1 and EMSA #2) of the 5hmC-enriched region. No significant protein-DNA binding was detected in the absence of 5hmC. ( b ) In the presence of 5hmC, distinct DNA-protein complexes were observed at both EMSA #1 and #2 (arrow). The addition of an anti-MeCP2 antibody induced a block-shift phenomenon (*), confirming specific MeCP2 binding to 5hmC-containing sequences.

Article Snippet: Primary antibodies against CREB (1:1000, Cell Signaling Technology, cat. no. 9192, rabbit polyclonal, Danvers, MA, USA) and MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) were used.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Blocking Assay

Journal: Acta Histochemica et Cytochemica

Article Title: Role of MeCP2 in Shaping the Histopathological Heterogeneity of Ampullary Carcinoma

doi: 10.1267/ahc.25-00049

Figure Lengend Snippet: Interaction between MeCP2 and CREB demonstrated by immunoprecipitation. Whole-cell lysates were immunoprecipitated with an anti-MeCP2 antibody, followed by western blotting with an anti-CREB antibody. A distinct band corresponding to CREB was detected at 43 kDa in the MeCP2 immunoprecipitates. No significant bands were observed in the input control (Input) or in immunoprecipitates with a nonspecific rabbit IgG (nR-IgG).

Article Snippet: Primary antibodies against CREB (1:1000, Cell Signaling Technology, cat. no. 9192, rabbit polyclonal, Danvers, MA, USA) and MeCP2 (1:1000, Cell Signaling Technology, cat. no. 3456, rabbit polyclonal, Danvers, MA, USA) were used.

Techniques: Immunoprecipitation, Western Blot, Control