Journal: Cellular & Molecular Biology Letters

Article Title: The novel tRNA-derived fragment, tiRNA-Met, inhibits the malignant progression of triple-negative breast cancer by regulating RANBP3L via a targeted interaction with SNRPA

doi: 10.1186/s11658-025-00738-2

Figure Lengend Snippet: tiRNA-Met directly interacted with SNRPA and promoted its ubiquitination. A , B Silver-stained SDS-PAGE gel images showed proteins immunoprecipitated by tiRNA-Met and its antisense RNA in MDA-MB-231 ( A ) and BT-549 ( B ) cell lines. C Western blot analysis of products from RNA pull-down assays using tiRNA-Met sense and antisense confirmed that tiRNA-Met directly associated with SNRPA. D , E RIP followed by RT-qPCR demonstrated specific binding of tiRNA-Met to SNRPA in MDA-MB-231 ( D ) and BT-549 ( E ) cells. F RNA pull-down and western blot assays revealed that tiRNA-Met bound to the RRM2 domain of SNRPA. G The mRNA expression levels of SNRPA remained unchanged in MDA-MB-231 and BT-549 cells transfected with tiRNA-Met mimics or inhibitor. H Overexpression of tiRNA-Met reduced SNRPA protein levels in both cell lines, while inhibition of tiRNA-Met increased SNRPA protein levels. I MDA-MB-231 cells treated with cycloheximide (Chx; 5 μM) and then transfected with tiRNA-Met mimics exhibited a reduced half-life of endogenous SNRPA protein, as shown by western blot; β-actin served as a loading control. J Treatment with MG132 (20 μg/ml) led to a progressive increase in SNRPA protein levels over time; β-actin was used as a reference. K Immunoprecipitation assays indicated that the binding of SNRPA to ubiquitinated proteins was elevated in MDA-MB-231 cells transfected with tiRNA-Met mimics. All data are presented as means ± SD; ns, P > 0.05; * P < 0.05; ** P < 0.01; **** P < 0.0001

Article Snippet: Biotin-labeled tiRNA-Met sense and antisense sequences were obtained from Genescript (Nanjing, China).

Techniques: Ubiquitin Proteomics, Staining, SDS Page, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Binding Assay, Expressing, Transfection, Over Expression, Inhibition, Control

Journal: iScience

Article Title: Divergent input patterns to the central lateral amygdala play a duet in fear memory formation

doi: 10.1016/j.isci.2024.110886

Figure Lengend Snippet: Modulation of CB1Rs in the LPB Glu →CeL SOM pathway affects conditioned fear memory acquisition (A) Schematic of the in vitro slice recording paradigm. (B) Representative average traces of eEPSCs in the absence (top) and presence of the CB1R agonist WIN55,212-2 (middle) or the CB1R antagonist AM251 (bottom). (C) Bath application of WIN55,212-2 significantly decreased the amplitude of eEPSCs, whereas AM251 significantly increased the amplitude of eEPSCs compared with the baseline (one-way ANOVA, F (2,12) = 57.97, ∗∗∗∗ p < 0.0001; Tukey’s post hoc test, Baseline versus WIN55, ∗∗∗ p = 0.0004; Baseline versus AM251, ∗∗∗ p = 0.0005; WIN55 versus AM251, ∗∗∗∗ p < 0.0001; n = 5 neurons from 3 mice for each group). (D) Schematic of the injection paradigm to test whether CB1Rs in the CeL control fear memory acquisition. (E) Mice with CeL injections of the CB1R agonist WIN55 exhibited similar fear responses across the duration of fear conditioning as the other two groups (two-way ANOVA, group: F (2,21) = 0.5255, p = 0.5988; time: F (3,63) = 93.78, ∗∗∗∗ p < 0.0001; interaction: F (6,63) = 0.824, p = 0.5556; n = 8 mice). (F and G) Microinjection of the agonist WIN55 into the CeL significantly decreased freezing levels in the contextual (F, one-way ANOVA, F (2,21) = 11.52, ∗∗∗ p = 0.0004; Tukey’s post hoc test, Vehicle versus WIN55, ∗∗∗ p = 0.0004; WIN55 versus WIN55+AM251, ∗ p = 0.0101; n = 8 mice) and cued (G, two-way ANOVA, group: F (2,21) = 2.892, p = 0.0777; time: F (1,21) = 214.3, ∗∗∗∗ p < 0.0001; interaction: F (2,21) = 5.214, ∗ p = 0.0145; Bonferroni’s post hoc test, freezing for Vehicle versus WIN55 during Tone presentation, ∗∗ p = 0.0014; n = 8 mice) fear memory tests, and the effect of WIN55 could be reversed with administration of the CB1R antagonist AM251. (H and I) The expression levels of CB1R mRNA in the LPB (H, two-sided unpaired t -test, t (1/10) = 4.282, ∗∗ p = 0.0016, n = 6 mice) and CB1R protein in the CeL (I, two-sided unpaired t -test, t (1/10) = 4.426, ∗∗ p = 0.0013, n = 6 mice) both decreased significantly after LPB injection of siRNA targeting CB1Rs. (J) Time course and schematic of the injection paradigm to identify the effects of CB1Rs at the LPB→CeL terminals on fear memory acquisition. (K) There were no significant differences among the groups in freezing levels during the fear conditioning session (two-way ANOVA, group: F (3,28) = 0.3493, p = 0.79; time: F (3,84) = 98.5, ∗∗∗∗ p < 0.0001; interaction: F (9,84) = 0.4148, p = 0.9239. n = 8 mice). (L and M) Effects of selective CB1R knockdown in the LPB on CB1R agonist-induced contextual (L, one-way ANOVA, F (3,28) = 8.272, ∗∗∗ p = 0.0004; Bonferroni’s post hoc test, NC + Vehicle versus NC + WIN55, ∗∗ p = 0.0011; NC + WIN55 versus siRNA+Vehicle, ∗∗ p = 0.0012; n = 8 mice) and cued (M, two-way ANOVA, F (3,28) = 12.8, ∗∗∗∗ p < 0.0001; Bonferroni’s post hoc test, before Tone presentation: For NC + Vehicle versus siRNA+Vehicle, ∗ p = 0.033; For NC + WIN55 versus siRNA+Vehicle, ∗∗∗ p = 0.0007; For siRNA +WIN55 versus siRNA+Vehicle, ∗ p = 0.011; during Tone presentation: For NC + Vehicle versus NC + WIN55, ∗∗∗ p = 0.0007; For siRNA+Vehicle versus NC + WIN55, ∗∗∗ p = 0.0003; n = 8 mice) fear memory impairment. All of the data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: SiRNA sequence : CB1R Sense : GACAUUCAGUACGAAGAUATT Antisense : UAUCUUCGUACUGAAUGUCTT , Genepharma , N/A.

Techniques: In Vitro, Injection, Control, Microinjection, Expressing, Knockdown

Journal: iScience

Article Title: Divergent input patterns to the central lateral amygdala play a duet in fear memory formation

doi: 10.1016/j.isci.2024.110886

Figure Lengend Snippet:

Article Snippet: SiRNA sequence : CB1R Sense : GACAUUCAGUACGAAGAUATT Antisense : UAUCUUCGUACUGAAUGUCTT , Genepharma , N/A.

Techniques: Virus, Recombinant, Protein Extraction, Purification, Sequencing, Negative Control, Software