Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A) Experimental design for (B)–(H). (B) Representative images of maximal projection and 3D reconstruction (IMARIS) of mG + astrocytes. (C and D) Surface area and enclosed volume of mG + astrocytes. Graphed as means of each astrocyte in Aldh1l1-CreER T2 :Sox2 fl/fl mice (left, n = 28 control, 43 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (E–H) Automatic tracing of mG + astrocyte processes by the filament tool of IMARIS and quantifications. Graphed as means of each astrocyte (left, n = 19 control, 17 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (I) Left, experimental design for (J)–(P); right, immunofluorescence of SOX2 and GFAP and quantification. n = 4 control, 4 Sox2 icKO. (J) Ctx astrocytes visualized by SR101 (arrows) for whole-cell recordings. (K) Voltage steps for astrocyte recording, from −180 to +20 mV with a step size of 10 mV. (L and M) Representative current tracing (L) and I-V curve (M) of one Sox2 -deficient and one Sox2 -intact astrocyte. (N1–N3) Quantification of cell capacitance (N1, n = 23 control, 18 Sox2 icKO), input resistance (N2, n = 20 control, 19 Sox2 icKO), and resting membrane potential (N3, n = 20 control, 17 Sox2 icKO) of Ctx astrocytes. (O) Western blot and quantification of Kir4.1 in the brain. n = 4 control, 4 Sox2 icKO. (P) Representative images and quantification of Kir4.1 intensity in the cortex. n = 4 control, 4 Sox2 icKO. Error bars indicate means ± SEM. Unpaired two-tailed Student’s t test was used for statistically analyzing two groups of data. Please see for statistics. n, biological replicates. Scale bars, (B, E, I, J, and P) 20 μm.

Article Snippet: Rabbit polyclonal anti-Kir4.1 , Alomone labs , Cat# APC-035; RRID: AB_2040120.

Techniques: Immunofluorescence, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Kir4.1 , Alomone labs , Cat# APC-035; RRID: AB_2040120.

Techniques: Protein Extraction, Recombinant, Electron Microscopy, Injection, Magnetic Cell Separation, Lysis, Bicinchoninic Acid Protein Assay, Western Blot, Glutamate Assay, Labeling, SYBR Green Assay, Chromatin Immunoprecipitation, Software, Activity Assay

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A) Experimental design for (B)–(H). (B) Representative images of maximal projection and 3D reconstruction (IMARIS) of mG + astrocytes. (C and D) Surface area and enclosed volume of mG + astrocytes. Graphed as means of each astrocyte in Aldh1l1-CreER T2 :Sox2 fl/fl mice (left, n = 28 control, 43 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (E–H) Automatic tracing of mG + astrocyte processes by the filament tool of IMARIS and quantifications. Graphed as means of each astrocyte (left, n = 19 control, 17 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (I) Left, experimental design for (J)–(P); right, immunofluorescence of SOX2 and GFAP and quantification. n = 4 control, 4 Sox2 icKO. (J) Ctx astrocytes visualized by SR101 (arrows) for whole-cell recordings. (K) Voltage steps for astrocyte recording, from −180 to +20 mV with a step size of 10 mV. (L and M) Representative current tracing (L) and I-V curve (M) of one Sox2 -deficient and one Sox2 -intact astrocyte. (N1–N3) Quantification of cell capacitance (N1, n = 23 control, 18 Sox2 icKO), input resistance (N2, n = 20 control, 19 Sox2 icKO), and resting membrane potential (N3, n = 20 control, 17 Sox2 icKO) of Ctx astrocytes. (O) Western blot and quantification of Kir4.1 in the brain. n = 4 control, 4 Sox2 icKO. (P) Representative images and quantification of Kir4.1 intensity in the cortex. n = 4 control, 4 Sox2 icKO. Error bars indicate means ± SEM. Unpaired two-tailed Student’s t test was used for statistically analyzing two groups of data. Please see for statistics. n, biological replicates. Scale bars, (B, E, I, J, and P) 20 μm.

Article Snippet: The following antibodies were used for our IHC study: SOX2 (1:500, Santa Cruz biotechnology); BLBP (1:200, Millpore); GFAP (1:500, Millpore); GFAP (1:500, Agilent); SOX9 (1:200, R&D system); Sox10 (1:300, Abcam); EYFP/GFP (1:500, abcam); Kir4.1 (1:400, Alomone labs); Sox9 (1:500, R&D system); VGlut1 (1:500, Millipore); PSD95 (1:500, Synaptic Systems); NeuN (1:500, Millipore); c-Fos (1:500, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used for our IHC study: SOX2 (1:500, Santa Cruz biotechnology); BLBP (1:200, Millpore); GFAP (1:500, Millpore); GFAP (1:500, Agilent); SOX9 (1:200, R&D system); Sox10 (1:300, Abcam); EYFP/GFP (1:500, abcam); Kir4.1 (1:400, Alomone labs); Sox9 (1:500, R&D system); VGlut1 (1:500, Millipore); PSD95 (1:500, Synaptic Systems); NeuN (1:500, Millipore); c-Fos (1:500, Santa Cruz Biotechnology).

Techniques: Protein Extraction, Recombinant, Electron Microscopy, Injection, Magnetic Cell Separation, Lysis, Bicinchoninic Acid Protein Assay, Western Blot, Glutamate Assay, Labeling, SYBR Green Assay, Chromatin Immunoprecipitation, Software, Activity Assay

Journal: PLoS ONE

Article Title: Slc26a7 Chloride Channel Activity and Localization in Mouse Reissner’s Membrane Epithelium

doi: 10.1371/journal.pone.0097191

Figure Lengend Snippet: Expression of key functional transport proteins that are known to be in the stria vascularis and that mediate generation of the endocochlear potential and the high potassium concentration of the luminal fluid, endolymph. There were no differences in the expression between Slc26a7 +/+ ( top panel; A, B, C ) and Slc26a7 Δ/Δ ( lower panel; D, E, F ) mice for the potassium channels Kcnq1 and Kcnj10 and for the Nkcc1 transporter Slc12a2 .

Article Snippet: The other primary antibodies used were goat anti-Kcnq1 1∶200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Kcnj10 1∶300 (Alomone, Jerusalem, Israel) and rabbit anti-Slc12a2 1∶100 (Chemicon, Temecula, CA) and incubated with appropriate secondary antibodies, as before .

Techniques: Expressing, Functional Assay, Concentration Assay

Journal: eBioMedicine

Article Title: Kir4.1 channel activation in NG2 glia contributes to remyelination in ischemic stroke

doi: 10.1016/j.ebiom.2022.104406

Figure Lengend Snippet: Deficits of Kir4.1 and MBP expression in both ischemic human patients and tMCAO mice. (a and b) Representative CT brain scans show an epileptic patient before and after the resective surgery (a) and an acute ischemic patient before and after decompressive craniectomy (b). The high density contrast area marked with red dotted line confirms the cerebral infarction of the ischemic stroke patient after decompressive craniectomy. (c) Representative images (left panel) and summary bar graph (right panel) of Kir4.1 and MBP expression levels from 10 non-ischemic surgical patients as comparison and 6 acute ischemic stroke patients, respectively. There is a significant decrease of Kir4.1 and MBP expression in ischemic cerebral tissues compared with that in non-ischemic patients. n = 6 and 10 patients for each group, respectively. ∗∗∗p < 0.001, two-tailed unpaired t-test. (d) The experimental diagram illustrates an established transient middle cerebral artery occlusion (tMCAO) mouse model of ischemic stroke. After MCA occlusion for 40 min, 2,3,5-triphenyltetrazolium chloride (TTC) staining was observed after 24 h reperfusion to detect the extent of the infarction in a series of brain sections. Representative images of Kir4.1 and MBP expression levels of ipsilateral infarction region by Western blottings at day 0, 1, 3, 6, and 9 of the onset of tMCAO. (e) The summary bar graphs show the expression changes of Kir4.1 and MBP in a 9-days time window of tMCAO. Note that both Kir4.1 and MBP show a continuous decrease starting at 24 h after reperfusion of tMCAO, while after the sixth day, they both rebounded synchronously. ∗∗∗p < 0.001. n = 4 mice for each group, one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests.

Article Snippet: The primary antibodies used for Western blotting were rabbit anti-Kir4.1 (Alomone Labs Cat# APC-035, RRID: AB_2040120), mouse anti-MBP (Millipore Cat# MAB382, RRID: AB_94971), and mouse anti-β-actin (Thermo Fisher Scientific Cat# MA5-15739, RRID: AB_10979409).

Techniques: Expressing, Two Tailed Test, Staining, Western Blot

Journal: eBioMedicine

Article Title: Kir4.1 channel activation in NG2 glia contributes to remyelination in ischemic stroke

doi: 10.1016/j.ebiom.2022.104406

Figure Lengend Snippet: The impairment of Kir4.1 channels expressed in NG2 glia leads to myelin loss in axons from tMCAO mice. (a and b) Representative traces show macroscopic currents and Ba 2+ -sensitive currents in NG2 glia in both contralateral (a) and ipsilateral (b) hippocampal CA1 regions of tMCAO. The arrow shows a dramatic loss of Ba 2+ -sensitive Kir4.1 current in ipsilateral NG2 glia after tMCAO. (c) Average I/V plot is for Ba 2+ -sensitive currents in NG2 glia after tMCAO. The error bars represent s.e.m. ∗∗∗p < 0.001, two-tailed unpaired t-test. n indicates the number of cells recorded. (d) Electron micrographs demonstrate the presence of impaired axons with demyelination in ipsilateral cortex compared with its contralateral region after 40 min tMCAO in wild type mice at postnatal 8 weeks. In contralateral cortex of tMCAO, axons show normal myelin, which exhibits dark, ring-shaped sheaths surrounding the axon. Scale bars: 2 μM. (e) Bar graph and G-ratio of myelinated axons represent the number of myelinated axons and G-ratio between ipsilateral cortex after tMCAO with its contralateral side. n indicates the number of axons from 4 wild type mice. (f) The box-plots represent average of myelin sheath thickness in both wild type mice and Kir4.1 deficient mice after tMCAO. The data were normally distributed and statistical significance was assessed within each group using Tukey–Kramer multiple comparisons test. ∗∗∗p < 0.001, n.s indicates not significant. The analyzed axons are from 4 mice per group. (g and h) Electron micrographs (g), bar graph and G-ratio of axonal diameter (h) show the demyelinated axons in both contralateral and ipsilateral hippocampus after 40 min tMCAO in Pdgfrα-CreER™;Kir4.1 f/f mice at postnatal 8 weeks. Scale bars: 2 μM. (i) The box-plots represent the average of G-ratio of myelinated axons in both wild type mice and Kir4.1 deficient mice after tMCAO. The data were normally distributed and statistical significance was assessed within each group using Tukey–Kramer multiple comparisons test. ∗∗p < 0.01, ∗∗∗p < 0.001, n.s indicates not significant. The analyzed axons are from 4 mice per group.

Article Snippet: The primary antibodies used for Western blotting were rabbit anti-Kir4.1 (Alomone Labs Cat# APC-035, RRID: AB_2040120), mouse anti-MBP (Millipore Cat# MAB382, RRID: AB_94971), and mouse anti-β-actin (Thermo Fisher Scientific Cat# MA5-15739, RRID: AB_10979409).

Techniques: Two Tailed Test

Journal: eBioMedicine

Article Title: Kir4.1 channel activation in NG2 glia contributes to remyelination in ischemic stroke

doi: 10.1016/j.ebiom.2022.104406

Figure Lengend Snippet: Deletion of Kir4.1 channels in astrocytes does not lead to myelin loss. (a) Representative images show a complete deletion of Kir4.1 (in red) in GFP (+) astrocytes (in green) of M1 region by injection of Cre-dependent AAV vector (AAV2/5-gfaABC1D-EGFP-P2A-iCre) into Kir4.1 f/f mouse cortex. (b) Electron micrographs demonstrate that there is no obvious alteration of myelinated axons between the contralateral (non-injection site) and ipsilateral (astrocytic Kir4.1 deletion site) side in wild type mice at postnatal 8 weeks. Scale bars: 2 μM. (c) Bar graph and G-ratio of myelinated axons represent the number of myelinated axons and G-ratio between ipsilateral M1 cortex with its contralateral side. n indicates the number of axons from 2 GFAP-GFP-Cre;Kir4.1 −/− mice. (d) The box-plot represents the average of myelin sheath thickness in contralateral and ipsilateral M1 region in GFAP-GFP-Cre;Kir4.1 −/− mice. The data were not normally distributed and statistical significance was assessed using Mann–Whitney unpaired test. n.s indicates not significant. The analyzed axons are from 2 mice.

Article Snippet: The primary antibodies used for Western blotting were rabbit anti-Kir4.1 (Alomone Labs Cat# APC-035, RRID: AB_2040120), mouse anti-MBP (Millipore Cat# MAB382, RRID: AB_94971), and mouse anti-β-actin (Thermo Fisher Scientific Cat# MA5-15739, RRID: AB_10979409).

Techniques: Injection, Plasmid Preparation, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Kir4.1 channel activation in NG2 glia contributes to remyelination in ischemic stroke

doi: 10.1016/j.ebiom.2022.104406

Figure Lengend Snippet: Luteolin specifically augments Kir4.1 channel currents in NG2 glia. (a) Representative traces show macroscopic current (in black), luteolin-induced macroscopic current (in magenta) and Ba 2+ -sensitive Kir4.1 currents after luteolin application (in blue) in NG2 glia from Pdgfrα-creER™;mGFP mice. (b) Bar graph summary showing the percentage of augmentation in Ba 2+ -sensitive Kir4.1 currents between control (basal) and luteolin application when the cell voltage was held at −150 mV and +40 mV, respectively. The error bars represent s.e.m. ∗∗∗p < 0.001, two-tailed unpaired t-test. n = 7 cells recorded for each group. (c) Representative traces show macroscopic current (in black), luteolin-induced macroscopic current (in magenta) and Ba 2+ -sensitive Kir4.1 currents after luteolin application (in blue) in NG2 glia from Pdgfrα-creER™; Kir4.1 −/− mice. Note that luteolin did not induce the augmentation of Kir4.1 current in NG2 glia in Kir4.1 cKO mice. (d) Bar graph summary showing the percentage of augmentation in Ba 2+ -sensitive Kir4.1 currents between control (basal) and luteolin application when the cell voltage was held at −150 mV and +40 mV, respectively. The error bars represent s.e.m. n.s indicates not significant, two-tailed unpaired t-test. n = 8 and 7 cells for control and luteolin group, respectively. (e) Representative traces show macroscopic current (in grey), luteolin-induced macroscopic current (in magenta) and Ba 2+ -sensitive Kir4.1 current after luteolin application (in blue) in astrocytes from wild type mice. (f) Bar graph summary showing the percentage of augmentation in Ba 2+ -sensitive Kir4.1 currents between control (basal) and luteolin application when the cell voltage was held at −150 mV and +40 mV, respectively. The error bars represent s.e.m. n.s indicates not significant, two-tailed unpaired t-test. n = 9 and 10 cells for control and luteolin group, respectively.

Article Snippet: The primary antibodies used for Western blotting were rabbit anti-Kir4.1 (Alomone Labs Cat# APC-035, RRID: AB_2040120), mouse anti-MBP (Millipore Cat# MAB382, RRID: AB_94971), and mouse anti-β-actin (Thermo Fisher Scientific Cat# MA5-15739, RRID: AB_10979409).

Techniques: Two Tailed Test

Journal: eBioMedicine

Article Title: Kir4.1 channel activation in NG2 glia contributes to remyelination in ischemic stroke

doi: 10.1016/j.ebiom.2022.104406

Figure Lengend Snippet: Luteolin treatment improves remyelination and Kir4.1 expression in tMCAO mice. (a and b) Representative electron micrographs show that the presence of impaired axons with demyelination in ipsilateral cortex compared with its contralateral region at day 9 after tMCAO in wild type mice saline group at postnatal 8 weeks (a) and the remyelinated axons in ipsilateral cortex compared with its contralateral region at day 9 after tMCAO in wild type mice after luteolin treatment at postnatal 8 weeks (b). Scale bars: 2 μM. (c and d) The percentage of healthy axons (the normal diameter of myelinated axons is over 200 nm) between contralateral and ipsilateral cortex after 9 days tMCAO in saline and luteolin group. n indicates the number of axons. The data were normally distributed and statistical significance was assessed within each group using two-tailed unpaired t-test. ∗∗p < 0.01; ∗∗∗p < 0.001, n.s indicates not significant. (e) The box-plots represent average of myelin sheath thickness between saline group and luteolin treatment group after 9 days tMCAO. The data were normally distributed and statistical significance was assessed within each group using two-tailed unpaired t-test. ∗∗∗p < 0.001, n.s indicates not significant. The analyzed axons are from 6 mice per group. (f) The box-plots represent average G-ratio of myelinated axons between saline group and luteolin treatment group after 9 days tMCAO. The data were normally distributed and statistical significance was assessed within each group using two-tailed unpaired t-test. ∗∗∗p < 0.001, n.s indicates not significant. The analyzed axons are from 6 mice per group. (g) Representative images of Kir4.1 and MBP protein expression in sham and tMCAO mice (contralateral and ipsilateral sides) after 9 days luteolin treatment. Note that both Kir4.1 and MBP expressions are significantly increased in ipsilateral sides after luteolin administration in tMCAO mice compared with its saline group. (h) Summary graphs show the average of Kir4.1 and MBP expressions by Western blot in sham mice and both contralateral and ipsilateral sides of tMCAO mice after 9 days luteolin treatment. ∗∗p < 0.01, n.s indicates not significant, n = 3 mice for both saline and luteolin group in sham control; n = 6 mice for both saline and luteolin group in contralateral side after tMCAO; n = 8 mice for both saline and luteolin group in ipsilateral side after tMCAO, one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests.

Article Snippet: The primary antibodies used for Western blotting were rabbit anti-Kir4.1 (Alomone Labs Cat# APC-035, RRID: AB_2040120), mouse anti-MBP (Millipore Cat# MAB382, RRID: AB_94971), and mouse anti-β-actin (Thermo Fisher Scientific Cat# MA5-15739, RRID: AB_10979409).

Techniques: Expressing, Two Tailed Test, Western Blot