Fig 1: Rasd2 overexpression in the NAcc attenuates depression-like behavior and activates the DRD2-cAMP-PKA-DARPP-32 signaling pathway in mice.a Schematic of experimental design and timeline. b Schematic diagram of microinjection of lentivirus vectors into mouse NAcc. Duration of immobility (c), climbing (d), and swimming (e), and the number of fecal boli (defecation) (f) in the forced swim test. N = 10, 11 mice in ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: duration of immobility, t(19) = 2.100, p = 0.0493; duration of climbing, t(19) = 2.130, p = 0.0465; duration of swimming, t(19) = 3.216, p = 0.0046; defecation, t(19) = 2.590, p = 0.0180. Representative tracks (g) and heatmaps (h) displaying mouse movement in the open field test. Total distance moves (i), line crosses (j), rearing (k), and center entries (l) in the open field test. N = 10, 11 mice for ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: distance, t(19) = 0.8095, p = 0.4282; line crosses, t(19) = 1.413, p = 0.1737; rearing, t(19) = 0.5033, p = 0.6206; center entries, t(19) = 0.5485, p = 0.5897. m Graph represents sucrose preference (%) before (Day 0) and after (Day 23) 5-d UMS. N = 10, 11 mice for ov-control and ov-Rasd2 groups, respectively. Two-way ANOVA (factor 1: time; factor 2: treatment): Ftime×treatment (1, 38) = 0.9972, p = 0.3243; Ftime (1, 38) = 21.83, p < 0.0001; Ftreatment (1,38) = 9.783, p = 0.0034. Tukey’s HSD post hoc test: after treatment, p(ov-control vs. ov-Rasd2) = 0.0288; in the ov-Rasd2 mice, p(baseline vs. test) = 0.0011. n Duration of immobility in the tail suspension test. N = 10, 11 mice in ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: t(19) = 3.015, p = 0.0071. o Rasd2 overexpression in the NAcc increased the GluA1 expression in the mPFC after 5-d UMS. N = 4, 5 mice in ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: t(7) = 2.391, p = 0.0481. p Rasd2 overexpression activated the DRD2-cAMP-PKA-DARPP-32 signaling pathway in the NAc. N = 4-5 mice. Two-tailed unpaired t-test: RASD2, t(8) = 4.255, p = 0.0028; DRD1, t(8) = 0.5450, p = 0.6006; DRD2, t(7) = 2.591, p = 0.0359; cAMP, t(7) = 2.692, p = 0.0310; PKA, t(7) = 3.887, p = 0.0060; DARPP-32, t(7) = 2.689, p = 0.0311. q Rasd2 overexpression increased the co-expression of RASD2 and DRD2 (scale bar, 20 μm). N = 4 mice per group. Two-tailed unpaired t-test: t(20) = 2.270, p = 0.0344. The data are expressed as mean ± s.e.m. *p < 0.05 and **p < 0.01. 5-d UMS 5-day unpredictable mild stress, OFT open field test, FST forced swimming test, TST tail suspension test, WB western blotting, IF immunofluorescence, GluA1 glutamate receptor subunit 1, DRD1 dopamine D1 receptor, DRD2 dopamine D2 receptor, cAMP cyclic adenosine monophosphate, PKA protein kinase A, DARPP-32 dopamine and cAMP-regulated phosphoprotein, 32 kDa, NAcc NAc core.
Fig 2: Rasd2 overexpression in DRD2PrL-NAcc neurons decreases depression-like behavior.a Schematic of experimental design and timeline. b Representative image for the identification of Drd2-cre genotypes. c Schematic diagram of microinjection of viral vectors into mouse PrL and NAcc sites (top, left); Representative images of EGFP (top, middle) and fDIO-mCherry (top, right) injection sites in the NAcc and PrL, respectively; Representative images of mCherry- (middle, left) and EGFP- (middle, middle) positive neurons in the PrL; Representative image of mCherry and EGFP co-labeled in the PrL (middle, right), arrows indicate co-expressed neurons.; Representative images of mCherry- (bottom, left) and EGFP- (bottom, middle) positive neurons in the NAcc; Representative image of mCherry and EGFP co-labeled in the NAcc (bottom, right). d Representative images showing co-localization of RASD2 and DRD2 in NAcc 3 weeks after virus injection (left, scale bar = 20 μm) and the number of DRD2-positive neurons that co-express RASD2 in NAcc (Right). N = 4 mice per group. Two-tailed unpaired t-test: t(22) = 2.101, p = 0.0474. Representative tracks (e) and heatmaps (f) displaying mouse movement in the open field test. Total distance moved (g), line crosses (h), rearing (i), and center entries (j) in the open field test. N = 10 mice per group. Two-tailed unpaired t-test: distance, t(18) = 0.9870, p = 0.3367; line crosses, t(18) = 0.0605, p = 0.9524; rearing, t(18) = 0.3031, p = 0.7653; center entries, t(18) = 0.8985, p = 0.3808. Duration of immobility (k), climbing (l), and swimming (m), and the number of fecal boli (defecation) (n) in the forced swim test. N = 10 mice per group. Two-tailed unpaired t-test: immobility, t(18) = 3.658, p = 0.0018; climbing, t(18) = 2.384, p = 0.0284; swimming, t(18) = 2.115, p = 0.0486; defecation, t(18) = 0.5153, p = 0.6128. o Graph represents sucrose preference (%) before (Day 0) and after (Day 23) treatment. N = 10 mice for each group. Two-way ANOVA (factor 1: time; factor 2: treatment): Ftime×treatment (1, 36) = 3.817, p = 0.0586; Ftime (1, 36) = 1.321, p = 0.2581; Ftreatment (1,36) = 3.817, p = 0.0586. Tukey’s HSD post hoc test: after treatment, p(WT vs. Drd2-cre) = 0.0426. p Duration of immobility in the tail suspension test. N = 10 mice per group. Two-tailed unpaired t-test: t(18) = 3.300, p = 0.0040. The data are expressed as mean ± s.e.m. *p < 0.05. 5-d UMS 5-day unpredictable mild stress, OFT open field test, FST forced swim test, TST tail suspension test, IF immunofluorescence, WT wild type, Cg1 cingulate cortex 1, PrL prelimbic cortex, IL infralimbic cortex, NAcc NAc core, DRD2 dopamine D2 receptor.
Fig 3: Activation of the PrL-NAcc circuit attenuates depression-like behavior after 5-d UMS.a Schematic of experimental design and timeline. b Schematic diagram of microinjection of viral vectors into mouse PrL and NAcc sites (top, left); Representative images of CaMKIIa-cre injection site in NAcc (green) (top, middle) and hM4Di injection site in PrL (red) (top, right); Representative images of mCherry- (middle, left) and EGFP- (middle, middle) positive neurons in the PrL; Representative image of mCherry and EGFP co-labeled in the PrL (middle, right), arrows indicate co-expressed neurons; Representative images of mCherry- (bottom, left) and EGFP- (bottom, middle) positive neurons in the NAcc; Representative image of mCherry and EGFP co-labeled in the NAcc (bottom, right). Scale bar = 200 μm, 100 μm and 20 μm. Representative tracks (c) and heatmaps (d) displaying mouse movement in the open field test. Total distance moved (e), line crosses (f), rearing (g), and center entries (h) in the open field test. N = 8 mice for each group. Two-way ANOVA (factor 1: CNO; factor 2: virus): distance, FCNO×virus (2, 42) = 0.4916, p = 0.6151; Fvirus (2, 42) = 0.4595, p = 0.6347; FCNO (1, 42) = 1.368, p = 0.2488; line crosses, FCNO×virus (2, 42) = 0.0273, p = 0.9731; Fvirus (2, 42) = 0.2017, p = 0.8182; FCNO (1, 42) = 1.571, p = 0.2170; rearing, FCNO×virus (2, 42) = 2.242, p = 0.1188; Fvirus (2, 42) = 0.4293, p = 0.6538; FCNO (1, 42) = 5.893, p = 0.0196; center entries, FCNO×virus (2, 42) = 0.4996, p = 0.6103; Fvirus (2, 42) = 0.2914, p = 0.7487; FCNO (1, 42) = 0.0937, p = 0.7611. Duration of immobility (i), climbing (j), and swimming (k), and the number of fecal boli (defecation) (l) in the forced swim test. N = 8 mice for each group. Two-way ANOVA (factor 1: CNO; factor 2: virus): immobility, FCNO×virus (2, 42) = 8.899, p = 0.0006; Fvirus (2, 42) = 2.824, p = 0.0707; FCNO (1, 42) = 0.0006, p = 0.9374. Tukey’s HSD post hoc test: in CNO-treated mice, p(control vs. hM3Dq) = 0.0287, p(hM3Dq vs. hM4Di) = 0.0031; in hM3Dq-treated mice, p(saline vs. CNO) = 0.0414; climbing, FCNO×virus (2, 42) = 5.285, p = 0.0090; Fvirus (2, 42) = 1.645, p = 0.2052; FCNO (1, 42) = 1.917, p = 0.1735. Tukey’s HSD post hoc test: in hM4Di-treated mice, p(saline vs. CNO) = 0.0199; swimming, FCNO×virus (2, 42) = 6.231, p = 0.0043; Fvirus (2, 42) = 4.903, p = 0.0122; FCNO (1, 42) = 14.91, p = 0.0004. Tukey’s HSD post hoc test: in CNO-treated mice, p(control vs. hM3Dq) = 0.0137, p(hM3Dq vs. hM4Di) = 0.0030; in hM3Dq-treated mice, p(saline vs. CNO) = 0.0001; defecation, FCNO×virus (2, 42) = 1.371, p = 0.2650; Fvirus (2, 42) = 0.2623, p = 0.7705; FCNO (1, 42) = 3.382, p = 0.0730. m Graph represents sucrose preference (%) before (Day 0) and after (Day 23) treatment. N = 8 mice for each group. Three-way ANOVA (factor 1: CNO; factor 2: virus; factor 3: time): FCNO×virus×time (2, 84) = 5.077, p = 0.008; Ftime×virus (2, 84) = 2.096, p = 0.129; Ftime×CNO (1, 84) = 0.426, p = 0.516; FCNO×virus (2, 84) = 4.261, p = 0.017; Fvirus (2, 84) = 2.101, p = 0.129; FCNO (1, 84) = 0.537, p = 0.466; Ftime (1, 84) = 3.529, p = 0.064. Tukey’s HSD post hoc test: in CNO + hM3Dq-treated mice, p(baseline vs. treatment) < 0.001; after treatment: in CNO-treated mice, p(control vs. hM3Dq) < 0.001, p(hM3Dq vs. hM4Di) < 0.001; in hM3Dq-treated mice, p(saline vs. CNO) < 0.001. n Duration of immobility in the tail suspension test. N = 8 mice for each group. Two-way ANOVA (factor 1: CNO; factor 2: virus): FCNO×virus (2, 42) = 1.706, p = 0.1940; Fvirus (2, 42) = 5.142, p = 0.0101; FCNO (1, 42) = 5.142, p = 0.0011. Tukey’s HSD post hoc test: in CNO-treated mice, p(control vs. hM3Dq) = 0.0212; in hM3Dq-treated mice, p(saline vs. CNO) = 0.0128. o Effects of PrL-NAcc circuit activation/inhibition on the activity of RASD2-positive neurons in the NAcc after 5-d UMS (scale bar, 20 μm). Two-way ANOVA (factor 1: CNO; factor 2: virus): FCNO×virus (2, 48) = 10.92, p = 0.0001; Fvirus (2, 48) = 8.605, p = 0.0006; FCNO (1, 48) = 0.03595, p = 0.8504. N = 3 mice per group. Tukey’s HSD post hoc test: in CNO-treated mice, p(control vs. hM3Dq) = 0.0329, p(control vs. hM4Di) = 0.0379, p(hM3Dq vs. hM4Di) < 0.0001; in hM3Dq-treated mice, p(saline vs. CNO) = 0.0100; in hM4Di-treated mice, p(saline vs. CNO) = 0.0450. The data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.001. 5-d UMS 5-day unpredictable mild stress, OFT open field test, FST forced swim test, TST tail suspension test, IF immunofluorescence, CNO clozapine-N-oxide, DRD2 dopamine D2 receptor, NAcc NAc core, Cg1 cingulate cortex 1, PrL prelimbic cortex, IL infralimbic cortex.
Fig 4: The mechanisms of 5-d UMS in inducing depression-like behavior involve RASD2 and DRD2.a The representative images showing RASD2 expression in the NAc (top, scale bar = 200 and 20 μm), and the number of RASD2-expressing neurons in NAcc and NAcs (bottom). N = 4 mice per group. Two-tailed unpaired t-test: NAcc, t(19) = 2.418, p = 0.0258; NAcs, t(20) = 2.591, p = 0.0175. b Representative images showing RASD2 expression in the mPFC (top, scale bar = 200 and 20 μm), and the number of RASD2-expressing neurons in Cg1, PrL, and IL (bottom). N = 3 mice per group. Two-tailed unpaired t-test: Cg1, t(10) = 2.721, p = 0.0215; PrL, t(11) = 0.9488, p = 0.3631; IL, t(11) = 3.407, p = 0.0059. c 5-d UMS caused changes in RASD2 and dopamine-related protein expression in the NAc. N = 4–7 mice. Two-tailed unpaired t-test: RASD2, t(7) = 3.050, p = 0.0186; DRD1, t(9) = 2.315, p = 0.0458; DRD2, t(7) = 2.597, p = 0.0356; DARPP-32, t(8) = 2.313, p = 0.0494; GluA1, t(10) = 1.035, p = 0.3251. d 5-d UMS caused changes in RASD2 and dopamine-related protein expression in the mPFC. N = 4–7 mice. Two-tailed unpaired t-test: RASD2, t(8) = 2.507, p = 0.0365; DRD1, t(10) = 0.4337, p = 0.6737; DRD2, t(8) = 0.9415, p = 0.3740; DARPP-32, t(7) = 2.981, p = 0.0205; GluA1, t(10) = 2.250, p = 0.0482. e Western blot images of a RASD2 immunoprecipitation showing co-immunoprecipitation of RASD2 and DRD2 in the NAc (left). Quantification of DRD2 co-immunoprecipitation with RASD2 immunoprecipitation. Each sample was normalized by its input and then DRD2 was normalized for the amount of RASD2 immunoprecipitated (right). N = 5 mice per group. Two-tailed unpaired t-test: t(8) = 6.616, p = 0.0002. f Schematic of experimental design and timeline. g 5-d UMS reduces the projection of PrL to the NAcc (scale bar, 20 μm). N = 4 mice per group. Two-tailed unpaired t-test: Cg1, t(22) = 1.665, p = 0.1102; PrL, t(22) = 2.202, p = 0.0385; IL, t(22) = 0.4018, p = 0.6917. The data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.01. 5-d UMS 5-day unpredictable mild stress, IF immunofluorescence, DRD1 dopamine D1 receptor, DRD2 dopamine D2 receptor, DARPP-32 dopamine and cAMP-regulated phosphoprotein, 32 kDa, GluA1 glutamate receptor subunit 1, FG fluorogold, NAcc NAc core, NAcs NAc shell, Cg1 cingulate cortex 1, PrL prelimbic cortex, IL infralimbic cortex.
Fig 5: Impact of RASD2 knockdown on thyroid cancer cell behavior. (A) Western blot validation of RASD2 knockdown efficiency using three different shRNAs (sh‐RASD2#1, #2, #3) in IHH4 and TPC‐1 cells. (B) Cell viability assessment by CCK‐8 assay in sh‐RASD2#3 and sh‐NC transfected cells at 24, 48, and 72 h. (C) EdU incorporation assay comparing proliferation between sh‐RASD2#3 and sh‐NC groups. (D) Transwell invasion assay results for sh‐RASD2#3 vs. sh‐NC groups (magnification: ×200). (E) Glycolytic parameters (glucose consumption, lactate production, ATP levels) in sh‐RASD2#3 and sh‐NC transfected IHH4 and TPC‐1 cells. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from Abcam for Anti-RASD2 antibody