Fig 2: Effect of TRIM47 knockdown in dendritic development. (A) Photomicrograph showing effective knockdown of TRIM47 protein level in hippocampal neurons (DIV 21) by two distinct shRNAs. (B) Quantitation of TRIM47 normalized protein level following knockdown. n = 3; *p < 0.006 and **p < 0.003; and an unpaired t-test with Welch’s correction. (C) Representative photomicrographs showing dendritic complexity (DIV 21) from hippocampal neurons transduced with lentivirus expressing two distinct shRNAs against TRIM47 (DIV 3). Dendritic development is analyzed by Sholl analysis. Scale as indicated. (D) Quantitation of Sholl analysis showing dendritic intersections at concentric circles drawn at 10 μm radius interval from the center of the soma. n = 30–32. One-way ANOVA with Dunnett’s T3 multiple comparisons test was used to measure the statistical significance. P < 0.92 (control RNAi vs. TRIM47 knockdown by shRNA#1) and p < 0.98 (control RNAi vs. TRIM47 knockdown by shRNA#2); and the Kolmogorov–Smirnov test. Data represents mean ± SEM.

Fig 3: Dendritic spine morphology following TRIM47 knockdown. (A) Representative photomicrograph showing dendritic spine morphology from hippocampal neurons (DIV 21) that are transduced with two shRNAs against TRIM47 (DIV 3) and transfected with chicken βactin promoter driven GFP to visualize dendritic spine. Scale as indicated. (B) Quantitation of the total dendritic spine density following TRIM47 knockdown. Spine density per micrometer of dendritic length is measured from confocal images using Neurolucida 360. ***p < 0.0009 for shRNA#1, **p < 0.0029 for shRNA#2. (C–E) Quantitation of thin spine (C), stubby spines (D), and mushroom spines (E) following TRIM47 knockdown. ***p < 0.0001 for (E), ns = not significant for (C,D). n = 16–25. One-way ANOVA with Dunnett’s T3 multiple comparisons test. Data are shown as the mean ± SEM.

Fig 4: Excitatory synapse formation following TRIM47 knockdown. (A) Synapse density is measured from hippocampal neurons after effective knockdown of TRIM47. Neurons (DIV 3) are transduced with two distinct shRNAs against TRIM47 and these neurons (DIV 21) are immunostained using presynaptic protein Synapsin I (magenta) as well as postsynaptic protein PSD95 (green). Synapses are visualized by apposing Synapsin I and PSD95 puncta onto hippocampal neurons expressing mCherry (red). PSD95/Synapsin I colocalized puncta per micrometer of dendrite is measured to detect synapse density. Scale 20 μm. (B) Quantitation of synapse density is measured from neurons expressing control shRNA (Control RNAi) and two distinct shRNAs (shRNA # 1 and shRNA# 2) against TRIM47. n = 15–21. ***p < 0.001; one-way ANOVA with Dunnett’s T3 multiple comparisons test. Data represent the mean ± SEM.

Fig 5: Enhancement of TRIM47 expression during spatial memory formation. (A) Schematic of the Spatial Object Recognition (SOR) memory test. (B) A representative heatmap showing the time spent by rat in the arena during the test session. (C) Quantitation of the average percentage time spent by rats exploring each object during the last training sessions and test sessions. Rats which were tested for object location memory where object location was same during the training and the test sessions (object not displaced or ND) and object location altered (object displaced or D) during test sessions. *p < 0.0114, **p < 0.0012; n = 4. Data are shown as the mean ± SEM, one-way ANOVA, and Fisher’s least significant difference (LSD). (D) Enhancement of TRIM47 in the hippocampus was detected from animals showing preferential exploration of the displaced object. A photomicrograph showing TRIM47 and Tuj1 proteins expression levels in tissue isolated from dorsal hippocampi of rats subjected to a spatial object recognition memory test. ND—rats subjected to SOR where the object location was same during the test and training sessions; D—rats subjected to SOR where the location of one of the objects was changed during the test session relative to the training sessions. (E) Quantitation of western blot analysis post SOR. *p < 0.0434; n = 4. An unpaired t-test with Welch’s correction is conducted. Data represent the mean ± SEM.