Journal: bioRxiv

Article Title: Reduced Kv3.1 Activity in Dentate Gyrus Parvalbumin Cells Induces Vulnerability to Depression

doi: 10.1101/784090

Figure Lengend Snippet: a . Thigmotaxis behavior in the open field test (OF) in WT (black dots, n= 13 mice) and p11 cKO (red dots, n= 13). *** p < 0.001 by unpaired t -test. b . Subthreshold social defeat stress (SSDS) included 3 defeat sessions. The social interaction test (SI) was conducted the next day. IZ, interaction zone. c . Ratio between the times spent in the IZ in the presence and absence of an unfamiliar aggressor in WT (n= 6) and p11 cKO mice (n= 6). ** p = 0.007 by unpaired t -test. Dashed line represents social avoidance threshold. d . Time spent in the IZ in the presence of an unfamiliar aggressor. ** p = 0.001 by unpaired t -test. e. Novel object recognition test (NOR), in WT (n= 15) and p11 cKO mice (n= 15). * p = 0.024 by unpaired t -test. f. OF thigmotaxis 3 weeks after DIO-DREADD virus injection to the DG in mCherry (n= 8), Gi-DREADD (n= 9), and Gs-DREADD (n= 9) mice. One Way ANOVA. F (2, 23) = 15.07, P <0.0001. *** p <0.0001 by post hoc Bonferroni. g. Ratios of time spent in the IZ in mCherry (n= 8), Gi-DREADD (n= 8), and Gs-DREADD (n= 7) treated mice. One Way ANOVA. F (2, 20) = 6.43, P =0.007. ** p = 0.004 by post hoc Bonferroni. h. Time spent in the IZ. One Way ANOVA. F (2, 20) = 9.21, P =0.002. * p =0.028 by post hoc Bonferroni. i. NOR in mCherry (n= 7), Gi-DREADD (n= 10), and Gs-DREADD (n= 10) mice. One Way ANOVA. F (2, 24) = 1.23, P = 0.319.

Article Snippet: 20 μg protein was loaded onto 4-12% BisTris gel, and transferred onto PVDF membranes that were incubated in 5% Milk in TBST and then with mouse anti-Kv3.1β (NeuroMab UC-DAVIS, 75-041, 1: 1,000), rabbit anti-HA (Cell Signaling, 3724 1: 1,000) rabbit anti-β-actin (Cell Signaling Technologies, 4970, 1: 2,000) or goat anti-p11 (R&D Systems, AF2377,1:200).

Techniques: Virus, Injection

Journal: bioRxiv

Article Title: Reduced Kv3.1 Activity in Dentate Gyrus Parvalbumin Cells Induces Vulnerability to Depression

doi: 10.1101/784090

Figure Lengend Snippet: a. Ratio between the time spent in the open and closed arms in the elevated plus maze test (EPM) in WT (n= 9) and cKO (n= 9). * p = 0.024 by unpaired t -test. b. Sucrose preference test (SPT) in WT mice and cKO (n= 11, 10), after 24 and 48 hrs. of consumption. Unpaired t -tests. c-e . Effect of 14 days of citalopram (CIT, 10 mg/kg, intraperitoneally) or vehicle (Veh, 100 μl saline) on depressive-like behaviors. c . Latency to approach food pellet in the novelty suppressed feeding test (NSF) in WT mice (n= 7 Veh, 7 CIT) and cKO (n= 7 Veh, 8 CIT). Two Way ANOVA. F Genotype X treatment (1, 25) = 5.8, P= 0.024. ** p =0.004 vs. WT Veh by post hoc Bonferroni. d. Immobility time in the forced swim test (FST) in WT (n= 7 Veh, 7 CIT) and cKO (n= 7 Veh, 7 CIT). Two Way ANOVA. F Genotype X treatment (1, 24) = 5.4, P= 0.030. * p = 0.040 vs. WT Veh by post hoc Bonferroni. e . Immobility time in the tail suspension test (TST) in WT (n= 8 Veh, 8 CIT) and cKO (n= 7 Veh, 8 CIT). Two Way ANOVA. F Genotype X treatment (1, 27) = 5.8, P= 0.023. * p =0.019 vs. WT Veh by post hoc Bonferroni. f . Exploration in the NOR in WT (n= 15) and p11 cKO (n= 15). p = 0.589 by unpaired t -test.

Article Snippet: 20 μg protein was loaded onto 4-12% BisTris gel, and transferred onto PVDF membranes that were incubated in 5% Milk in TBST and then with mouse anti-Kv3.1β (NeuroMab UC-DAVIS, 75-041, 1: 1,000), rabbit anti-HA (Cell Signaling, 3724 1: 1,000) rabbit anti-β-actin (Cell Signaling Technologies, 4970, 1: 2,000) or goat anti-p11 (R&D Systems, AF2377,1:200).

Techniques: Saline, Suspension

Journal: bioRxiv

Article Title: Reduced Kv3.1 Activity in Dentate Gyrus Parvalbumin Cells Induces Vulnerability to Depression

doi: 10.1101/784090

Figure Lengend Snippet: a. Bar graph summary of mRNA expression levels of the HCN isoform genes in PV hippocampal neurons from p11 WT PV TRAP mice. b , c . Representative traces of HCN currents ( b ) and the I-V curves ( c ) showing the HCN amplitude in PV neurons from WT and cKO mice in response to increasing 10 mV potential steps from −140 mV to −50 mV (n = 5/ 3 for WT and 3/ 2 for cKO). Unpaired t -tests.

Article Snippet: 20 μg protein was loaded onto 4-12% BisTris gel, and transferred onto PVDF membranes that were incubated in 5% Milk in TBST and then with mouse anti-Kv3.1β (NeuroMab UC-DAVIS, 75-041, 1: 1,000), rabbit anti-HA (Cell Signaling, 3724 1: 1,000) rabbit anti-β-actin (Cell Signaling Technologies, 4970, 1: 2,000) or goat anti-p11 (R&D Systems, AF2377,1:200).

Techniques: Expressing

Journal: bioRxiv

Article Title: Reduced Kv3.1 Activity in Dentate Gyrus Parvalbumin Cells Induces Vulnerability to Depression

doi: 10.1101/784090

Figure Lengend Snippet: a . Representative immunoblot from N2A cell lysates after 48 hrs. of transient co-transfection of Kv3.1β with p11 or without (control). Arrows and numbers represent protein weights in KDa. b . Immunoblot from N2A lysates after 48 hrs. of co-transfection of KV3.1α with p11 or without (control). c . Densitometry of the protein levels in n=5 wells per group. * p = 0.031, ** p = 0.001 by unpaired t -test. d . Immunoblot from representative lysates of N2a cell lines: Untransfected (control) or stably transfected with Kv3.1β-GFP. e . Cells were transfected with siRNA for p11 or control (siRNA-scr.) and lysed after 24 hrs. f. Densitometry of the protein levels in siRNA-p11 (n= 4 wells) and siRNA-scr (n= 4 wells). * p = 0.039, *** p < 0.001 by unpaired t -test. g . Representative immunocytochemical images depicting co-localization between GFP and ATPase 1A1 (left), GM130 (right) and calnexin (bottom) in fixed Kv3.1β-GFP N2A cells transfected with either siRNA-scr. or siRNA-p11 for 24 hrs. Scale Bar, 5 μm. h. Co-localization ratio between GFP and the organelle markers. Dots represent individual cells and numbers inside the bars indicate the numbers of inspected cells. *** p < 0.001 by unpaired t -test.

Article Snippet: 20 μg protein was loaded onto 4-12% BisTris gel, and transferred onto PVDF membranes that were incubated in 5% Milk in TBST and then with mouse anti-Kv3.1β (NeuroMab UC-DAVIS, 75-041, 1: 1,000), rabbit anti-HA (Cell Signaling, 3724 1: 1,000) rabbit anti-β-actin (Cell Signaling Technologies, 4970, 1: 2,000) or goat anti-p11 (R&D Systems, AF2377,1:200).

Techniques: Western Blot, Cotransfection, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Reduced Kv3.1 Activity in Dentate Gyrus Parvalbumin Cells Induces Vulnerability to Depression

doi: 10.1101/784090

Figure Lengend Snippet: a . Representative immunohistochemical images showing Kv3.1β immunolabeling in DG PV cells from control mouse or in PV-Cre mouse after overexpression (O/E) of Kv3.1β. Scale bar, 50 μm. b . OF thigmotaxis after O/E of either GFP or Kv3.1β in DG PV cells, in PV-Cre (control, n= 11 GFP, and n= 9 Kv.3.1β) and p11 cKO (n= 8 GFP and n= 8 Kv.3.1β). Two Way ANOVA. F Genotype X AAV (1, 32) = 11.21; P= 0.0021, Genotype F = 11.78, P= 0.017; AAV F = 19.53, P= 0.001. *** p < 0.001 by post hoc Bonferroni. c . Ratios of time spent in the IZ after CSDS in PV-Cre mice with O/E of GFP (n= 10) or Kv3.1β (n= 8) in DG PV cells. * p= 0.034 by unpaired t -test. d . Time spent in the IZ. * p= 0.037 by unpaired t -test. e . WT mice were treated for 3 days with RE1 (0-100 μM in 100μl intraperitoneally) or with saline (control, n= 5 mice per group). OF thigmotaxis was determined 30 min after the third injection. One Way ANOVA. F (6, 28) = 4.1, P= 0.043. * p < 0.05 vs 0 μM by post hoc Bonferroni. f-h . Stress sensitive mice (IZ ratio ≤ 1) were identified in SI test (SI1) after CSDS, and were treated for 3 days with saline (100 μl intraperitoneally) and for additional 3 days either with RE1 (500 nM, 100 μl intraperitoneally) or Veh. The second SI test (SI2) was conducted 30 min after the last injection. g . Ratios of time spent in the IZ in n= 8 mice per group. * p= 0.049 by unpaired t -test. h. Time spent in the IZ in n= 8 mice per group. * p = 0.046 by unpaired t -test.

Article Snippet: 20 μg protein was loaded onto 4-12% BisTris gel, and transferred onto PVDF membranes that were incubated in 5% Milk in TBST and then with mouse anti-Kv3.1β (NeuroMab UC-DAVIS, 75-041, 1: 1,000), rabbit anti-HA (Cell Signaling, 3724 1: 1,000) rabbit anti-β-actin (Cell Signaling Technologies, 4970, 1: 2,000) or goat anti-p11 (R&D Systems, AF2377,1:200).

Techniques: Immunohistochemical staining, Immunolabeling, Over Expression, Saline, Injection

Journal: Breast Cancer Research

Article Title: Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity

doi: 10.1186/bcr1647

Figure Lengend Snippet: Plasminogen activation system component changes on PMA-treated MCF-7 cells. MCF-7 cells were cultured for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA. (a) Cells were washed and incubated for 30 minutes on ice with 10 to 20 μg/ml of anti-uPAR monoclonal antibody (mAb), anti-uPA mAb, anti-tPA mAb, anti-annexin II (Ann II) C-16 polyclonal antibody (pAb), anti-S100A10 (p11) pAb, or a matched isotype control antibody. Cells were then washed and incubated for 45 minutes on ice in the dark with a 1:200 dilution of appropriate secondary antibody labelled with fluorescein isothiocyanate (FITC). All cells were then washed and analysed for cell-surface-associated FITC fluorescence in the presence of 5 μg/ml propidium iodide using dual-colour flow cytometry. Specific antibody binding was calculated by subtracting control antibody fluorescence. Solid line indicates a fold increase of 1 (equivalent to no change). P values indicate a significant increase compared to the corresponding untreated control. (b) Confocal microscopy on attached cells was also used to verify enhancement of cell-surface uPAR by PMA treatment. PMA, 12-O-tetradecanoylphorbol 13-acetate; tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor.

Article Snippet: Goat anti-S100A10 (p11) mAb (AF1698) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Activation Assay, Cell Culture, Incubation, Control, Fluorescence, Flow Cytometry, Binding Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: Flow cytometry analysis of immune and glial cells in a trigeminal neuralgia rat model

doi: 10.1038/s41598-021-02911-x

Figure Lengend Snippet: Immunofluorescence staining of A1- and A2-reactive astrocytes in the TREZ on POD 28. ( a – c ) and ( g – i ) Immunofluorescence staining of reactive astrocytes in the TREZ in the two groups. ( d , j ) show GFAP/C3-positive A1 astrocytes in the TREZ. ( c , k ) show GFAP/S100A10-positive A2 astrocytes in the TREZ. ( f , l ) show triple immunofluorescence staining of GFAP, C3 and S100A10. ( d1 – f1 ) and ( j1 – l1 ) show the higher magnification of ( d – f ) and ( j – l ). ( a – l ). scale bar = 100 μm. ( d1 – f1 , j1 – l1 ), scale bar = 50 μm.

Article Snippet: Then, 1 ml of 1 × Perm/Wash Buffer (BD Pharmingen, NJ, USA) was added, and the cells were washed twice by centrifugation at 300 × g for 5 min. After resuspension in 100 μL of 1 × Perm/Wash buffer, the cells were incubated with 0.2 μg/mL anti-S100A10-Alexa Fluor 647 antibody (FAB2377R, R&D Systems, MN, USA) and 0.1 μg/mL anti-C3 antibody (ab17456, Abcam, Cambridge, UK) at room temperature for 30 min. Then, the cells were centrifuged at 400 × g for 5 min and resuspended in ice-cold PBS.

Techniques: Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Flow cytometry analysis of immune and glial cells in a trigeminal neuralgia rat model

doi: 10.1038/s41598-021-02911-x

Figure Lengend Snippet: Quantitative analysis of C3 and S100A10 expression area in GFAP positive astrocytes in TREZ on POD 28. ( a ) Quantitative analysis showing significant up regulation of C3 in GFAP positive astrocytes on POD 28 in TN group compared to sham group. ( b ) Quantitative analysis showing significant lower expression of S100A10 in GFAP positive astrocytes on POD 28 in TN group compared to sham group. ( c ) The ratio of GFAP/C3-positive area and GFAP/S100A10-positive area was clearly increased in TN group compared with sham group. Data represent mean ± s.e.m. (n = 4–5 per group). * p < 0.05, as determined two-tailed Student’s t-tests.

Article Snippet: Then, 1 ml of 1 × Perm/Wash Buffer (BD Pharmingen, NJ, USA) was added, and the cells were washed twice by centrifugation at 300 × g for 5 min. After resuspension in 100 μL of 1 × Perm/Wash buffer, the cells were incubated with 0.2 μg/mL anti-S100A10-Alexa Fluor 647 antibody (FAB2377R, R&D Systems, MN, USA) and 0.1 μg/mL anti-C3 antibody (ab17456, Abcam, Cambridge, UK) at room temperature for 30 min. Then, the cells were centrifuged at 400 × g for 5 min and resuspended in ice-cold PBS.

Techniques: Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: Flow cytometry analysis of immune and glial cells in a trigeminal neuralgia rat model

doi: 10.1038/s41598-021-02911-x

Figure Lengend Snippet: The proportions of different types of astrocytes in the TREZ and TG. ( a ) Flow contour plot of mature astrocytes (CD45 − GLAST + ), A1 astrocytes (CD45 − GLAST + C3 + ) and A2 astrocytes (CD45 − GLAST + S100A10 + ) in the TREZ and TG in the two groups of rats. ( b – d ) The difference between mature astrocytes (CD45 − GLAST + ) and A2 astrocytes (CD45 − GLAST + S100A10 + ) in the TN group and sham group was not statistically significant. Compared with the sham group, the proportion of A1 astrocytes (CD45 − GLAST + C3 + ) in the TN group showed a downward trend ( p = 0.1599). ns: not significant, as determined by two-tailed Student’s t-test.

Article Snippet: Then, 1 ml of 1 × Perm/Wash Buffer (BD Pharmingen, NJ, USA) was added, and the cells were washed twice by centrifugation at 300 × g for 5 min. After resuspension in 100 μL of 1 × Perm/Wash buffer, the cells were incubated with 0.2 μg/mL anti-S100A10-Alexa Fluor 647 antibody (FAB2377R, R&D Systems, MN, USA) and 0.1 μg/mL anti-C3 antibody (ab17456, Abcam, Cambridge, UK) at room temperature for 30 min. Then, the cells were centrifuged at 400 × g for 5 min and resuspended in ice-cold PBS.

Techniques: Two Tailed Test

Journal: Gut Microbes

Article Title: Gut microbial ammonia as a mediator of PFOS neurotoxicity and its remediation by the flavonoid Icaritin

doi: 10.1080/19490976.2026.2620125

Figure Lengend Snippet: ICT alleviates PFOS-induced activation of hippocampal astrocytes and Aβ pathology, neuroinflammation, and oxidative stress. (A) Representative H&E staining of the hippocampal dentate gyrus (DG) region showing neuronal morphology. Scale bars: 500 μm (upper panel, overview), 50 μm (lower panel, detailed view). (B) Dual immunofluorescence staining of GFAP (green, astrocyte marker) and Aβ (red) in the hippocampal DG region. (C) Quantitative analysis of GFAP fluorescence intensity ( n = 5). (D) Quantitative analysis of Aβ fluorescence intensity ( n = 5). (E) Triple-label immunofluorescence staining of GFAP (green), C3 (red) and S100A10 (purple) in the hippocampal DG region. (F) Quantitative analysis of C3 + GFAP + cells (%) ( n = 5). (G) Quantitative analysis of S100A10 + GFAP + cells (%) ( n = 5). (H−J) Hippocampal levels of pro-inflammatory cytokines: (H) IL-1β ( n = 6), (I) IL-6 ( n = 6), and (J) TNF- α ( n = 6), measured by ELIZA. (K) Reactive oxygen species (ROS) levels in hippocampal tissues ( n = 6). (L) Malondialdehyde (MDA) content, a lipid peroxidation marker ( n = 6). (M−O) Activities of antioxidant enzymes: (M) catalase (CAT, n = 6), (N) superoxide dismutase (SOD, n = 6), and (O) glutathione peroxidase (GSH-Px, n = 6) in hippocampal tissues. The data were presented as the mean ± SEM. ** P < 0.01 vs. Control group; # P < 0.05, ## P < 0.01 vs. PFOS group.

Article Snippet: GFAP (16825-1-AP), C3 (21337-1-AP), S100A10 (11250-1-AP) and β -tubulin (10094-1-AP) were purchased from Proteintech (Wuhan, China). β -amyloid (Aβ 1-42 ) peptide was purchased from Macklin (Shanghai, China).

Techniques: Activation Assay, Staining, Immunofluorescence, Marker, Fluorescence, Control