EMBO/soda-nel huggingface dataset:
Robotic platform for microinjection into single cells in brain tissue (2019)
Figure 4

Figure 4: Neural stem cell lineage tracing and expression of exogenous mRNA.

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 (A - G) or Dx-A488 along with mRNA for RFP (H - I), without (0 h, A, D-G) or with slice culture for 24 or 48 h (24 h, B, D-G, H, I; 48 h, C, D-G). After fixation slices were stained for Tbr2 and TuJI. (A-C) Fluorescence images of tissue after microinjection with dextran-A555 (magenta). The tissue was stained for Tbr2 (green) and TuJI (white).

A. Microinjected cell at 0 h; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2.

B. Two-daughter cell progeny 24 h after microinjection; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2; the bottom cell is a newborn BP positive for Tbr2. Both cells are negative for the neuronal marker TuJI (white).

C. Four-daughter cell progeny 48 h after microinjection; note that daughter 1 and 2 reside in the VZ, while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ.

D. Distribution of microinjected cells and their progeny in the VZ, SVZ and CP (cortical plate). n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.

E. Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface (0 µm) and expressed as % of total. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.

F. Expression of Tbr2 in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.

G. Expression of TuJI in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.

H. Organotypic slices of mouse E14.5 dorsal telencephalon injected with Dextran-A488 (green) and RFP poly-A+-mRNA. After 24h in culture the progeny expresses RFP (magenta; blue: DAPI, n = 17 cells injected, 8 RFP positive).

I. Percentage of RFP-positive and RFP-negative cells (n = 17 cells injected).

Data information: In (A-C and H) the images on the left are maximum intensity projections (MIP) of 27, 50 , 47 and 60-focal planes, respectively; images on the right are single optical sections corresponding to the nucleus area; scale bars are 20 µm for MIP and 10 µm for single focal planes. In (H), the scale bar in the top left image is 100 μm. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.
Robotic platform for microinjection into single cells in brain tissue (2019)
Figure 5

Figure 5: Quantitative dissection of cell-to-cell-communication in the developing brain.

Automated microinjection was performed on organotypic slices of mouse E14.5 - E15.5 dorsal telencephalon using a solution containing Dextran-A555 (not shown) and Alexa488 (green). Slices were fixed after microinjection (n = 71 cells total) and were stained for Tbr2 (white, H-J). (A-C) Representative examples of a non-coupled cell (A, cartoon on the right), a 2-cell cluster (B, cartoon on the right) and a 5-cell cluster (C, cartoon on the right). The asterisks indicate the Dx-A555-positive, microinjected cell.

D. Percentage of microinjected cells found in a coupled cluster (coupled, green).

E. Cluster size, expressed as % of total.

F. Distribution of coupled cells expressed as the distance from the ventricular surface (0 µm = ventricle surface.

G. Distribution of coupled cells divided into 11 bins and expressed as % of total.

H. A representative picture of a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2. Microinjected cells are stained for Tbr2.

H, I. A representative picture of (H) a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2) and (I) a 7-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 7), the latter containing one Tbr2 positive cell. Microinjected cells are stained for Tbr2. The asterisks indicate the Dx-A555-positive, microinjected cell.

J. comparison of Tbr2-positive cells (expressed as % of total) among all the cells in the VZ (VZ; n=1003 from 4 different confocal images from 3 independent experiments; error bar represents standard deviation) and in coupled clusters in the VZ (coupled clusters, VZ; n= 104 from two independent experiments).

K. Coupled cells were scored based one presence/absence of apical and/or basal polarity cues, and results are expressed as % of total.

Data information: In (A-C, H and I) the images on the left are maximum intensity projections (MIP) of 18, 17, 23, 12 and 30-focal planes, respectively; in (H and I), the images on the right are single optical sections corresponding to the nucleus (white dotted line); scale bars are 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.
Robotic platform for microinjection into single cells in brain tissue (2019)
Figure 6

Figure 6: Automated microinjection platform can be applied in a generalized manner to target neurons in the developing mouse and human telencephalon.

Automated microinjection of mouse (A-F) and human (G,H) neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 or human 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, C, D, H). Slices were fixed after microinjection (C, D-F, H) and stained for Reelin (white, C) and SatB2 (white, D).

A. Schematic of automated microinjection into neurons.

B. Phase contrast image of automated microinjection into mouse neurons. Scale bar is 100 µm.

C. Left: overview image of a mouse microinjected area (arrow indicates a pyramidal neuron, arrowhead indicates a Cajal-Retzius neuron; image is MIP of 20 focal planes). Microinjected neurons show different morphology and positivity for the Cajal-Retzius marker Reelin (white). Right, top row: the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin. Right, bottom row: the Cajal-Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin. Images on the top and bottom row left (LY MIP) are a MIP of 2 and 3 focal planes, respectively. The three images on the top and bottom right (LY, Reelin and DAPI) are single optical sections. Scale bar is 20 µm for MIP and 10 µm for single focal planes.

D. Overview image of a mouse microinjected area. The arrow indicates a pyramidal neuron; the image is MIP of 30 focal planes, scale bar: 50 µm. Row on the right: the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper-layers marker SatB2 (white). Images are single focal planes. Scale bar is 10 µm.

E. Proportion of neurons positive or negative for Reelin (n = 12 cells total).

F. Proportion of neurons positive or negative for SatB2 (n = 39 cells in total).

G. Schematic of human tissue hand slicing and microinjection.

H. Microinjected neurons in the developing human telencephalon. Top, MIP of 20 focal planes (scale bar: 50µm). Bottom: high magnification (scale bar: 10 µm).

Data information: VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.

Expanded View Figure Legends
Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system (2016)
Figure 1

Figure 1. The LTG inhibitor Bulgecin A prevents T6SS function.

A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ∆tssM cells or ∆tssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.

B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.
Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system (2016)
Figure 2

Figure 2. The MltE LTG is required for T6SS function.

Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 cells of the indicated strains. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB and HcpFL were immunodetected using anti-TolB (upper panel) and anti-FLAG (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.
Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system (2016)
Figure 3

Figure 3. The MltE peptidoglycan hydrolase activity is required for T6SS function.

A. Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 WT, ∆mltE cells or ∆mltE cells producing wild-type (mltE+) or E64Q mutant (mltEE64Q) VSV-G-tagged MltE (MltEV) from arabinose-inducible plasmids. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB, HcpFL and MltEV were immunodetected using anti-TolB (upper panel), anti-FLAG (middle panel) and anti-VSV-G (lower panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.

B. Anti-bacterial activity. E. coli K-12 prey cells (W3110 gfp+, kanR) were mixed with the indicated attacker cells, spotted onto Sci-1 inducing medium (SIM) agar plates and incubated for 4 hours at 37°C. The image of a representative bacterial spot and the average and standard deviation (n=3) of the relative fluorescence of the bacterial mixture (in arbitrary unit, AU) are shown in the upper graph. The number of recovered E. coli prey cells (counted on selective kanamycin medium) is indicated in the lower graph (in log10 of colony-forming unit (cfu)). The black, dark grey and light grey circles indicate values from three independent assays, and the average is indicated by the bar. The experiment was performed in triplicate and a representative result is shown. Asterisks indicate significant differences compared to the wild-type attacker strain (NS, non significant; ***, p < 0.001).
Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system (2016)
Figure 4

Figure 4. MltE interacts with the TssM periplasmic domain.

A-B. Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal indicator plates. The blue color of the colony reflects the interaction between the two proteins. TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins. The experiment was performed in triplicate and a representative result is shown.

C. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.
Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system (2016)
Figure 6

Figure 6. MltE is required for TssM multimerization.

A-B. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 EAEC wild-type or ∆mltE cells producing FLAG-tagged TssM (FLTssM) and/or HA-tagged TssJ (TssJHA, panel A) or TssL (TssLHA, panel B) (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM) and anti-HA (lower panel, TssJ or TssL) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiments were performed in triplicate and a representative result is shown.

C. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.

D. Fluorescence microscopy. Recordings showing TssM localization using the chromosomally-encoded sfGFP-tssM fusion in wild-type (WT) or ∆mltE cells or ∆mltE cells producing the wild-type (mltE+) or catalytic variant (mltEE64Q) MltE protein. Scale bars are 1 μm. The experiment was performed in triplicate and a representative result is shown.
SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function (2020)
Figure 1

Figure 1. Suppressed NMDAR-mediated synaptic transmission and plasticity in the PtenΔC/ΔC hippocampus.

(A) Normal basal excitatory synaptic transmission at SC-CA1 synapses of PtenΔC/ΔC mice (4-5 weeks), indicated by the input-output curve of the amplitudes of field excitatory postsynaptic potentials (fEPSPs) plotted against stimulus intensities. (n = 10 slices from 4 mice for WT and 9 (4) for ΔC, genotype P=0.8378, two-way repeated measures ANOVA with Bonferroni's multiple comparison test). The error bars represent Standard error of the mean SEM (see Appendix table S1 "Statistics" for full details.

(B) Normal paired-pulse facilitation at SC-CA1 synapses of PtenΔC/ΔC mice (4-5 weeks), indicated by fEPSP slope ratios plotted against inter-stimulus intervals. (n = 11 slices from 4 mice for WT and 11 (4) for ΔC, genotype P=0.7376, two-way repeated measures ANOVA with Bonferroni's test). The error bars represent SEM.

(C,D) Suppressed HFS-LTP and normal TBS-LTP at SC-CA1 synapses of PtenΔC/ΔC mice (4-6 weeks). The grey traces represent baseline fEPSP prior to LTP induction. The error bars represent SEM. (n = 9 slices from 6 mice for WT and 7 (6) for ΔC for HFS-LTP, and 14 (9) for WT and 8 (5) for ΔC for TBS-LTP, *P < 0.05, ns, not significant, Mann-Whitney U test and Student's t-test).

(E) Suppressed LFS-LTD at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). (n = 10 slices from 6 mice for WT and ΔC, **P < 0.01, Student's t-test). The grey traces represent baseline fEPSP prior to LTD induction. The error bars represent SEM.

(F) Normal mGluR-LTD at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). (n = 6 slices from 5 mice for WT and 8 (5) for ΔC, ns, not significant, Mann-Whitney U test). The grey traces represent baseline fEPSP prior to LTD induction. The error bars represent SEM.

(G) Reduced NMDA/AMPA ratio at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). The NMDAR component was obtained 60 ms after stimulation. (ratio, n = 11 cells from 8 mice for WT and 8 (8) for ΔC; decay, n = 12 cells from 8 mice for WT and 11 (8) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test). The error bars represent SEM.

(H) Reduced amplitude but not frequency of NMDA-mEPSCs at PtenΔC/ΔC CA1 pyramidal neurons. (n = 17 cells from 4 mice for WT and 18 (4) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM.

(I) Reduced NMDA/AMPA ratio at synapses in layer II/III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC/ΔC mice (P20-21). The NMDAR component was obtained 60 ms after stimulation. (n = 9 cells from 3 mice for WT and 10 (3) for ΔC, *P < 0.05, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.

Data information: (C-F) Results from the last 10-minute recordings are shown in scatter plots.
SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function (2020)
Figure 2

Figure 2. Increased expression of the Slc6a20a glycine transporter gene and decreased brain glycine levels in the PtenΔC/ΔC mice

(A) A volcano plot showing DEGs (adjusted p value < 0.1) derived from RNA-Seq results for PtenΔC/ΔC and WT mice (P21). For the calculation of adjusted p value, we have employed the R package DESeq2 where the P‐values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. (n = 3 mice for WT and ΔC). See also Dataset EV1 for the full RNA-Seq results.

(B) GSEA analysis of RNA-Seq results from PtenΔC/ΔC and WT mice (P21). Transcripts ranked by levels of expression (ΔC/WT ratio) were tested for enrichment in precurated gene sets in the C5 (gene ontology) category. NES, normalized enrichment score; positive and negative enrichments indicate a greater contribution of up- and downregulated genes, respectively, to the enrichments (see also Dataset EV2for the full GSEA results).

(C) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC/ΔC mice (P21). We denoted the antibody as 'SLC6A20' here, not 'SLC6A20A', because this antibody was generated using a synthetic peptide sequence (YNEPSNNCQKHAI) that is commonly present in SLC6A20A and SLC6A20B, being a pan-SLC6A20 antibody (ThermoFisher). (n = 3 mice for WT and ∆C, *P < 0.05, Student's t-test). The error bars represent SEM.

(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.

(F) Increased binding of β-catenin in the promoter region of the Slc6a20a gene in PtenΔC/ΔC mice (P21), revealed by ChIP (chromatin immunoprecipitation) assay. Note that β-catenin binding was increased selectively in the target region for the primer set #4 indicated in the diagram. TSS, transcriptional start site; P1-4; promoter regions targeted by the four primer sets. (n = 7 mice for WT-P4 and ΔC-P4 and 3 mice for WT-P1/2/3 and ΔC-P1/2/3, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.
SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function (2020)
Figure 3

Figure 3. Decreased extracellular proline and glycine levels in the PtenΔC/ΔC brain and increased climbing in juvenile and adult PtenΔC/ΔC mice.

(A) Normal proline but decreased whole-brain glycine levels in the brains of PtenΔC/ΔC mice (P21), as determined by ELISA analysis of the S1 fraction, obtained after the removal of unbroken cells and large debris (see Materials and Methods for details). (proline, n = 10 mice for WT and 8 for ΔC, glycine, n = 9 mice for WT and 8 for ΔC, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.

(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.

(D) Increased repetitive climbing in juvenile and adult PtenΔC/ΔC mice at P30 (juvenile) and 2-4 months (adult), as indicated by frequency and time spent climbing in Laboras cages, where mouse movements were continuously monitored for 72 hours. Shaded and unshaded periods; 12-hour light-off and light-on periods over 72 hours. (n = 22 mice for WT and 18 mice for ΔC for P30, and 25 for WT and 27 for ΔC for 2-4 months, *P < 0.05, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM.
SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function (2020)
Figure 5

Figure 5. Knockdown of SLC6A20A expression by antisense oligonucleotide increases whole-brain glycine levels and NMDAR function and rescues repetitive climbing in PtenΔC/ΔC mice.

(A) Schematic representation for the intracerebroventricular (ICV) injection of Slc6a20a antisense oligonucleotide (ASO) into the lateral ventricle (LV) for the reduction of Slc6a20a expression in the whole mouse brain.

(B) Slc6a20a-ASO decreases the levels of Slc6a20a mRNAs in the brain of WT and PtenΔC/ΔC mice (P20-32), as shown by qRT-PCR analysis performed 1-2 weeks after injection. (n = 5 mice for WT-Sal/saline, 4 for WT-ASO, 3 for ΔC-Sal, and 3 for ΔC-ASO, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test for each comparison). The error bars represent SEM.

(C) Slc6a20a-ASO decreases the levels of SLC6A20 proteins in the brain of WT mice (2-4 months), as shown by immunoblot analysis of whole-brain lysates six days after injection. (n = 4 mice, **P < 0.01, Student's t-test). The error bars represent SEM.

(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.

(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.

(F) PtenΔC/ΔC mice (P19-37) treated with Slc6a20a-ASO display unaltered mEPSC frequency and amplitude in CA1 pyramidal neurons, as compared with ASO-untreated PtenΔC/ΔC mice. Note that mEPSCs in WT mice are not affected by Slc6a20a-ASO treatment. (n = 13 neurons from 3 mice for WT-saline, 14 (2) for WT-ASO, 14 (3) for ΔC-saline, and 12 (3) for ΔC-ASO, ***P < 0.001, ns, not significant, two-way ANOVA with Bonferroni's test). The error bars represent SEM.

(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.

(H) Slc6a20a-ASO treatment has no effect on the self-grooming of PtenΔC/ΔC mice (2-4 months) in the Laboras test. (n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, ns, not significant, two-way ANOVA with Bonferroni's test). The error bars represent SEM.