Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations

doi: 10.1073/pnas.1716892115

Figure Lengend Snippet: ATM or ATR loss causes synaptic deficiency. (A) Cultured cortical neurons from WT or Atm KO mice were immunolabeled with anti-Bassoon (green) and anti-MAP2 (red) antibodies. (Scale bar: 20 μm.) (B) Quantification of Bassoon puncta in WT and Atm KO cortical neurons. n = 13–14 neurons from three batches of neuronal cultures. Error bars represent SEM. ****P < 0.0001, unpaired t test. (C) Representative images and quantification results of FM4-64 dye spontaneous release from neurites of WT (black) and Atm KO (red) cultures. (Scale bar: 20 μm.) n = 102–200 individual measurements from four independent neuron cultures. Error bars represent SEM. Significance of the difference between the curves, *P < 0.05 (multiple t tests). (D) Confocal images of WT cortical neurons stained with anti-VAMP2 (red), anti-ATM [2C1(1A1)] (green), or anti-ATR (red) antibodies. (Scale bar: 20 μm.) (E) Atr knockdown (red) with shRNA. n = 40–60 individual measurements from four separate neuronal cultures. Error bars represent SEM. Significance of the difference between the curves, *P < 0.05 (multiple t tests). (F) Scrambled (sc) or Atr-shRNA (red) transfected cortical neurons immunolabeled with anti-Bassoon (green) antibody. (Scale bar: 20 μm.) (G) Quantification of Bassoon (presynaptic puncta) density in control and Atr knockdown neurons. n = 17∼34 neurons from three different neuronal cultures. Error bars represent SEM. P = 0.0707, unpaired t test.

Article Snippet: For siRNA and ORF plasmids, Atr -siRNA (sc-29764) was obtained from Santa Cruz Biotechnology, GFP- Atm -shRNA (TL320267) and GFP- Atr -shRNA (TL519184) were supplied by Origene, mCherry- Atm -shRNA (MSH026597) was obtained from Genecopoeia, GFP-C1-PLCdelta-PH (21179) was provided by Addgene.

Techniques: Cell Culture, Immunolabeling, Staining, shRNA, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations

doi: 10.1073/pnas.1716892115

Figure Lengend Snippet: ATM and ATR protein levels show a reciprocal relationship. (A) Brain sections from 2-mo-old WT, Atmtm1Awb/Atmtm1Awb (Awb), and Atmtm1Bal/Atmtm1Bal (Bal) mice were labeled with ATR (green) or MAP2 (red) antibodies and counterstained with DAPI (blue). (Scale bars: as marked.) (B) ATR immunostaining intensity. n = 3 animals for each group. Error bars represent SEM. *P = 0.0232; *P = 0.036, unpaired t test. (C) Western blots of Atm KO (Awb) and WT cortical lysates. Actin served as a loading control. n = 3 animals for each group. Error bars represent SEM. ***P = 0.0006, unpaired t test. (D) Western blots of HEK293T cells lysates obtained at 48 h after transfection with Atm-shRNA or Atr-shRNA. GAPDH served as a loading control. (E) Quantification of the blots shown in D. n = 4–6 independent cultures. Error bars represent SEM. ***P = 0.0007; *P = 0.01; *P = 0.0445; **P = 0.0032, unpaired t test. (F) Western blots of ATM, ATR, VGLUT1, and VGAT of lysates from 21 DIV cortical neurons treated with D-CPP-ene or bicuculline for 24 h. (G) Quantification of the blots shown in F. n = 3∼4 independent neuronal cultures. Error bars represent SEM. For ATM, *P = 0.0317, *P = 0.0152; for VGLUT1, *P = 0.03; for ATR, **P = 0.0046, *P = 0.0213; for VGAT, *P = 0.0293, *P = 0.0479, unpaired t test.

Article Snippet: For siRNA and ORF plasmids, Atr -siRNA (sc-29764) was obtained from Santa Cruz Biotechnology, GFP- Atm -shRNA (TL320267) and GFP- Atr -shRNA (TL519184) were supplied by Origene, mCherry- Atm -shRNA (MSH026597) was obtained from Genecopoeia, GFP-C1-PLCdelta-PH (21179) was provided by Addgene.

Techniques: Labeling, Immunostaining, Western Blot, Transfection, shRNA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations

doi: 10.1073/pnas.1716892115

Figure Lengend Snippet: ATM or ATR deficiency leads to excitatory/inhibitory (E/I) imbalance. (A) 21 DIV cortical neurons isolated from Bal mice—heterozygous (Atm+/−), homozygous (Atm−/−), and WT (Atm+/+)— were stained with the indicated antibodies. (Scale bar: 100 μm.) (B) Percentages of VGLUT1+ and VGAT+ neurons in these cultures. n = 8–10 coverslips of neurons from four independent cultures. Error bars represent SEM. ns, P = 0.0512; **P = 0.0024; ***P = 0.0001; ****P < 0.0001, unpaired t test. (C) Representative confocal images of WT and Atm KO cortical neurons, stained with MAP2 (red), VGLUT1 (green), or VGAT (green) antibodies. (Scale bar: 20 μm.) High-magnification images show VGLUT1 or VGAT puncta on MAP2+ neurites. (D) Quantification of VGLUT1 and VGAT puncta in C. n = 15–23 neurons from four independent cultures. Error bars represent SEM. ****P < 0.0001, unpaired t test. (E) Atm or scrambled shRNA-transfected neurons immunolabeled by anti-VGLUT1 (red) and anti-VGAT (green) antibodies. (Scale bar: 50 μm.) (F) Ratio of VGLUT1 to VGAT protein intensity in E. n = 18–23 neurons from four independent cultures. Error bars represent SEM. ****P < 0.0001, unpaired t test. (G) Confocal images of scrambled or Atr shRNA (red)-transfected neurons, stained with anti-VGLUT1 (green) or anti-VGAT (green). (Scale bar: 20 μm.) High-magnification images show VGLUT1 or VGAT puncta on neurites. (H) Quantification of VGLUT1 and VGAT puncta in G. n = 17–19 neurons from three independent cultures. Error bars represent SEM. ns, P = 0.7341; **P = 0.0041, unpaired t test. (I) Atr or scrambled shRNA-transfected neurons immunolabeled with VGLUT1 (red) and VGAT (green) antibodies. (Scale bar: 50 μm.) (J) Ratio of VGLUT1 to VGAT protein intensity in I. n = 29–43 neurons from four independent cultures. Error bar represents SEM. ****P < 0.0001, unpaired t test.

Article Snippet: For siRNA and ORF plasmids, Atr -siRNA (sc-29764) was obtained from Santa Cruz Biotechnology, GFP- Atm -shRNA (TL320267) and GFP- Atr -shRNA (TL519184) were supplied by Origene, mCherry- Atm -shRNA (MSH026597) was obtained from Genecopoeia, GFP-C1-PLCdelta-PH (21179) was provided by Addgene.

Techniques: Isolation, Staining, shRNA, Transfection, Immunolabeling

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet: Synaptotagmin 2 is significantly upregulated in CamiCre + forebrain (A) qPCR analysis of mRNA expression in the hippocampus in indicated mouse lines. Syt2-Int8 represents pre-mRNA of Syt2 . Values were normalized by Gapdh expression level and are shown as fold increases against Cre - controls. Pgk1 is another housekeeping gene. (B and C) Western blotting images of different brain regions. CB, cerebellum; CC, cerebral cortex; HP, hippocampus; MB, midbrain; MO, medulla oblongata; OB, olfactory bulb. (D) Quantification results of C. Values were normalized against α-tubulin expression level and are shown as fold increases against Cre - controls. (E) Western blotting images of hippocampal lysates showing Syt2 expression levels in various Cre-expressing methodologies. Viral concentration was diluted 2x in three steps for AAV-hSyn-Cre expression. ∗, nonspecific band. Mean ± SD is shown. ∗∗p < 0.01; ∗∗∗p < 0.001. N = 4 (biological replicates). Statistical tests were performed using two-tailed, unpaired t-tests. See also Figure S2 .

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Expressing, Western Blot, Concentration Assay, Two Tailed Test

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet: Synaptotagmin 2 is ectopically overexpressed in the hippocampus, amygdala, cortical amygdala, and piriform cortex in CamiCre + brain Immunofluorescent images of Syt2 or VGlut1 staining in brains of CamiCre mice. The white dotted boxes in the left panels are magnified in the right panels. The magenta dotted boxes are areas used for colocalization analyses in Figure 3 . Amg, amygdala; Co-Amg, cortical amygdala; Hp, Hippocampus; Mo, motor cortex; Pir, piriform cortex. Scale bars: left 1 mm; right 250 μm.

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Staining

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of CamiCre + cerebrum (A) Representative high-resolution immunofluorescence images from corresponding areas indicated by magenta dotted boxes in Figure 2 sp, stratum pyramidale; sl, stratum lucidum. (B) Results from colocalization analyses on a total of 6.3 × 10 4 μm 2 per region. Ectopically expressed Syt2 showed significant colocalization with VGlut1 in CamiCre + brain. R, Pearson correlation coefficient; M1 and M2, Manders coefficient. NS, not significant; ∗∗∗p < 0.0001 (significant correlation or colocalization); †††p < 0.0001 (significant noncorrelation or noncolocalization compared to random displacement images). Scale bars: (CA1) 5 μm; (others) 10 μm.

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Immunofluorescence

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet: BAC transgene integration sites are within the Syt2 locus on chromosome one of CamiCre + mice (A) Results of TLA sequence coverage across the mouse genome. Chromosomes are indicated on the y axis, and the chromosomal position is shown on the x axis. The identified integration site is encircled in green. The coverage peak on chromosome 18 is owing to the homology of the BAC transgene with the endogenous genome. (B) Break points for BAC transgene integration sites were identified by TLA sequencing. Coding and noncoding regions in exons are indicated by indigo and light blue boxes, respectively. Red arrowheads indicate the three integration sites at positions 134,666,219, 134,668,306, and 134,669,891 of mouse chromosome 1 (GRC m38). See also Figure S4 .

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Sequencing

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet:

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Virus, Recombinant, Western Blot, Stripping, Immunodetection, Plasmid Preparation, Isolation, Sequencing, Software