mouse anti fascin 1  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1‐Induced Synaptic Plasticity in the Medial Prefrontal Cortex

doi: 10.1002/advs.202405352

Figure Lengend Snippet: Inhibition of morphine addiction memory after Fscn1 interference. A) Schematic diagram of microinjection into mPFC and brain atlas of mPFC. Bar = 500 µm. B) Fluorescent localization of mPFC in mouse. Bar = 40 µm. C,D) Decrease of Fscn1 mRNA and protein expression in mPFC of mice after Fscn1 knockdown (n = 6). The data were presented as the mean ± SEM; Unpaired t test; *** p < 0.001, **** p < 0.0001. E) CPP score of pre‐test (n = 8). The data were presented as the mean ± SEM; Two‐way ANOVA and Bonferroni's multiple comparisons test. F) CPP score of post‐test 1 (n = 8). The data were presented as the mean ± SEM; Two‐way ANOVA and Bonferroni's multiple comparisons test. **** p < 0.0001. G) CPP score of post‐test 2 (n = 8). The data were presented as the mean ± SEM; Two‐way ANOVA and Bonferroni's multiple comparisons test. * p < 0.05. # p < 0.05. H) Trajectory maps, trajectory heat maps and trajectory maps in 3D of Ctrl + Saline and Fscn1‐shRNA + Saline or Ctrl + Morphine and Fscn1‐shRNA + Morphine group during the pre‐test, post‐test 1 and post‐test 2. I–K) Other behavioral parameters, such as mean speed, total distance and zone transition number were not significantly different among different groups and test (n = 8). The data were presented as the mean ± SEM; Two‐way ANOVA and Bonferroni's multiple comparisons test.

Article Snippet: The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).

Techniques: Inhibition, Microinjection, Expressing, Knockdown, Saline, shRNA

Journal: Advanced Science

Article Title: The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1‐Induced Synaptic Plasticity in the Medial Prefrontal Cortex

doi: 10.1002/advs.202405352

Figure Lengend Snippet: Fscn1 knockdown resulted in loss of spines in the mPFC. A) Golgi‐stained mPFC sections which were extracted after post‐test on Day 17, scale bar = 1 mm. B,C) Representative basal dendrites of Ctrl and Fscn1‐shRNA mice. Bar = 25 µm. D,E) Representative apical dendrites from Ctrl and Fscn1‐shRNA mice; thin, stubby and mushroom‐type spines were showed by arrows. Bar = 20 µm. F) The representative magnified parts of dendrites taken from Ctrl mouse and Fscn1‐shRNA mouse. Bar = 10 µm. G) A decreased spine density was apparent in Fscn1‐shRNA mice (n = 10). The data were presented as the mean ± SEM. Unpaired t test. **** p < 0.0001. H) No difference was found in thin, stubby, mushroom spine fraction between Ctrl and Fscn1‐shRNA mice (n = 8). The data were presented as the mean ± SEM. Two‐way ANOVA and Bonferroni's multiple comparisons test. I–L) Fscn1‐shRNA mice loss of spines: including both thin and stubby ‐type spines were primarily observed on apical dendrites that were 100–140 µm from the soma of pyramidal cells (n = 8). The data were presented as the mean ± SEM. Two‐way ANOVA and Bonferroni's multiple comparisons test. * p < 0.05, ** p < 0.01, **** p < 0.0001. No difference was observed on mushroom‐type spines.

Article Snippet: The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).

Techniques: Knockdown, Staining, shRNA

Journal: Advanced Science

Article Title: The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1‐Induced Synaptic Plasticity in the Medial Prefrontal Cortex

doi: 10.1002/advs.202405352

Figure Lengend Snippet: Knockdown of Kdm7a/ Fscn1 blocked morphine‐induced elevation of neuronal activity in the mPFC. A) Virus localization and expression in the mPFC, which were extracted after post‐test on Day 17. Bar1 = 500 µm. Bar2 = 80 µm. B) Diagram of fiber photometry for recording RGECO1a activity from mPFC neurons before and after morphine‐induced CPP. Calcium‐dependent (560 nm) and calcium‐independent (410 nm) fluorescence signals were recorded. C) ΔF/F of calcium signals from Ctrl + Saline group (red line), Ctrl + Morphine (blue line), Kdm7a‐shRNA + Morphine (green line) and Fscn1‐shRNA + Morphine (purple line) neurons were recorded before (2 s) and after entry into the drug‐paired chamber (6 s) during pre‐test, post‐test 1 and post‐test 2. Heatmaps illustrating Ca 2+ signals aligned to the initiation of trials during pre‐test, post‐test 1 and post‐test 2. Each row plots one trial, and a total of 30 trials per group were illustrated. The color scale on the right indicates ΔF/F. D–F) The mean signal before (2 s) and after entry into the drug‐paired chamber (6 s) during pre‐test, post‐test 1 and post‐test 2 (n = 3). The data were presented as the mean ± SEM; One‐way ANOVA and Bonferroni's multiple comparisons test; ** p < 0.01, *** p < 0.001, **** p < 0.0001. ΔF/F = (Signal560 nm – Signal410 nm)/ Signal410nm.

Article Snippet: The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).

Techniques: Knockdown, Activity Assay, Virus, Expressing, Fluorescence, Saline, shRNA

Journal: Advanced Science

Article Title: The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1‐Induced Synaptic Plasticity in the Medial Prefrontal Cortex

doi: 10.1002/advs.202405352

Figure Lengend Snippet: KDM7A regulates Fscn1 expression via H3K9me2 and H3K27me2. A) Transfection of N2a cells with Kdm7a‐specific siRNA/ Exp, the cells were seeded at 50% confluency and transfected with siRNA / Exp duplexes at a final concentration of 50 nM. ChIP‐qPCR and Western blotting were performed after 48 h of transfection. B) N2a cells were visualized under an inverted microscope. C, E, G, I) The transfection of Kdm7a siRNA/ Exp resulted in significant downregulation/ upregulation of Kdm7a mRNA and protein expression in N2a cells (n = 6). The data were presented as the mean ± SEM; t test; * p < 0.05, ** P < 0.01, **** p < 0.0001. D, F, H, J) Kdm7a siRNA/ Exp treatment led to reduction/ growth in Fscn1 mRNA and protein expression in N2a cells (n = 6). The data were presented as the mean ± SEM; t test; ** p < 0.01, *** p < 0.001, **** p < 0.0001. K) The potential binding sites of H3K9me2 and H3K27me2 at Fscn1 promoter region from the Cistrome Data Browser database, the enrichment index is 0.13. L–N) The ChIP‐qPCR analysis was performed to assess the impact of Kdm7a siRNA and NC siRNA at 3 peak regions of Fscn1 promoter (n = 6). The data were presented as the mean ± SEM; Two‐way ANOVA and Bonferroni's multiple comparisons test. **** p < 0.0001. O–Q) The ChIP‐qPCR analysis was performed to assess the impact of Kdm7a‐Exp and NC‐Exp at 3 peak regions of Fscn1 promoter (n = 6). The data were presented as the mean ± SEM. Two‐way ANOVA and Bonferroni's multiple comparisons test. ** p < 0.01. **** p < 0.0001.

Article Snippet: The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).

Techniques: Expressing, Transfection, Concentration Assay, ChIP-qPCR, Western Blot, Inverted Microscopy, Binding Assay

Journal: Advanced Science

Article Title: The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1‐Induced Synaptic Plasticity in the Medial Prefrontal Cortex

doi: 10.1002/advs.202405352

Figure Lengend Snippet: Mechanistic hypothesis diagram. KDM7A targets Fscn1 to regulate synaptic plasticity by removing H3K9me2 and H3K27me2, which serves as a key regulator of morphine‐induced reward memory.

Article Snippet: The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).

Techniques:

Journal: Advanced Science

Article Title: The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1‐Induced Synaptic Plasticity in the Medial Prefrontal Cortex

doi: 10.1002/advs.202405352

Figure Lengend Snippet: Primer sequences for qRT‐PCR.

Article Snippet: The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).

Techniques: Sequencing