EMBO/SourceData huggingface dataset:
Figure 3Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD) .Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S3A-D, one-way ANOVA followed by Tukey's multiple comparison test.
Figure 4Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, T, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing TFM-4AS-1-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S4A-D, Tukey's multiple comparison test.
Figure 6A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected.B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo.D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9).E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8).
Figure 7A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison.C) Significance values from GSEA using combined STAT3 gene sets identified in B. Each treatment group is compared to tumor-bearing control (cachexia) transcriptomes.D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes.Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D.G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).
Figure 8A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis.C) Significance values from GSEA using combined CTNNB1 gene sets. Each treatment group was compared to tumor-free control transcriptomes. *FDR < 1e-5 determined, set to 1e-5 for plot.D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).
Figure 1A) Neutrophils and lymphocytes count.B) Levels of creatine kinase (CK) and creatinine.C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).D) Levels of erythrocyte sedimentation rate (ESR) and ferritin.E) Levels of D-dimer and lactate dehydrogenase (LDH).F) Proportion of total CD3+ and CD4+ T cells.G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio.H) Proportion of B and natural killer (NK) cells.cytokine profile of COVID-19 patients for IL-2, IL-4 (I)cytokine profile of COVID-19 patients for , IL-6, IL-10 (J) and TNF-α and IFN-γ (K).
Figure 2Time from symptom onset to initial diagnosis (TFSD).   Time from symptom onset to development of pneumonia (TFSP).   Time from symptom onset to treatment (TFST).Time from development of pneumonia to recovery (TFPR).
Figure 3Correlation between IL-6 and maximal body temperature during hospitalization.Correlation between IL-6 and C-reactive protein (CRP).Correlation between IL-6 and lactate dehydrogenase (LDH).Correlation between IL-6 and D-dimer.Correlation between IL-6 and ferritin.Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.
Figure 4IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right).IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition.The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.
Figure 5A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset.B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease.Both abnormal C-reactive protein (CRP) (D) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.
Figure 1RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 hours. Protein lysates were treated with λ Phosphatase (PPase) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=6 biological replicates).Control- or IRE1-siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).MEF cells were transfected with either empty vector, EGFP-FMRP or 3xFLAG-IRE1 plasmids then pre-treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG-18 (25 µM; 1 hour) followed by TG (600 nM) stimulation for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP, IRE1 and FMRP (n=3 biological replicates) and with LC-MS/MS. Identified IRE1 kinase-mediated FMRP phosphorylation sites (bottom).
Figure 2Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).    Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).  RCT experiment (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).
Figure 3A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).
Figure 4RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).
Figure 5Lesion area calculated from en face aorta, stained with ORO (n=12-13 mice per group; Scale bar = 5 mm).Lesion area calculated from en face aorta, stained with ORO (n=9 mice per group; Scale bar = 5 mm).
Figure 6Lesion area calculated from en face aorta, stained with ORO (n=5 mice per group; Scale bar = 5 mm).Lesion area calculated from en face aorta, stained with ORO (n=6 mice per group; Scale bar = 5 mm).
Figure 1(C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.
Figure 2Neutralization assays using VSVΔG pseudotyped with wildtype SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. (A) Sb#15, Sb#68, or an equimolar mixure of both sybodies. (B) Sb#14, Sb#16, Sb#45, or, as a positive control, the previously-described sybody MR3. (C) Neutralization by bivalent sybody-Fc fusions. Error bars correspond to standard deviations of three biological replicates.
Figure 4(C) Neutralization assay using VSVΔG pseudotyped with wildtype Spike-protein. Relative infectivity was determined in response to increasing GS4 or Tripod-GS4r concentration. The corresponding neutralization data for isolated Sb#15 and Sb#68 are provided as reference. (D) Control experiment using VSVΔG pseudotyped with VSV-G. Infectivity was not affected by Tripod-GS4r.
Figure 5(C) Neutralization assay using VSVΔG pseudotyped with Spike-protein containing the Q493R or P384H mutation, respectively. Relative infectivity in response to increasing binder concentrations was determined.
Figure 6(A) GCI-based kinetic analysis of interactions between immobilized sybodies (indicated above each plot) and wild-type (WT) or mutant RBDs carrying the individual K417N, E484K or N501Y mutations (left panels), the combined triple KEN (Beta) mutations (middle panels), or the double L452R/T478K (Delta) mutations (right panels). Sb#15, Sb#68 and GS4 were immobilized on independent flow-cells via biotinylated Avi-tags, and the RBD variants were sequentially injected at a concentration of 200 nM.
Figure 2A. Growth curves of the library (black) compared to two different controls under increasing selective pressures. DSB (double-stranded break) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK. n=3 for each curve
Figure 3B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3
Figure 4D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samplesE. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds
Figure 5C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samplesF. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples
Figure 3A Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding EGFP or EGFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. EGFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. B Number of EGFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to EGFP. Data are presented as mean ± S.E.M., n (EGFP +Cre) = 8 embryos; n (EGFP) = 8 embryos.C Representative image of E18.5 coronal brain sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding mRFP or mRFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. mRFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. D Number of mRFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to mRFP. Data are presented as mean ± S.E.M., n (mRFP+Cre) = 5 embryos; n (mRFP) = 5 embryos. E Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos co-electroporated in utero at E14.5 with plasmids encoding a fluorescent marker (green) together and Cre+KCC2FL, Cre+KCC2CTD, or Cre+KCC2R952H. DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. F Number of transfected neurons migrating above (II-IV, left) and below (V-VI/IZ-SVZ, right) the upper border of layer V in embryos electroporated with constructs in (E) normalized to respective data from embryos electroporated with Cre. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 to Cre. Data are presented as mean ± S.E.M., n (Cre+KCC2FL) = 13 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos.
Figure 4A, B, C The layer thickness and neuronal number within each layer was assessed using layer-specific markers in Kcc2-/-and Kcc2+/+ embryos at E18.5. Cux1 was used to label layers II-IV (A), Ctip2 to label layer V (B), and Tbr1 to label layer VI (C). Dashed white lines in the representative images in (A-C) indicate upper and lower layer boundaries. Sp, subplate. Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc (neuronal numbers) and one-way ANOVA with Holm-Sidak's post hoc (layer thickness). Data are presented as mean ± S.E.M., n = 11 embryos for both genotypes. Scale bar = 50 µm.D Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) in Kcc2-/- and Kcc2+/+ embryos electroporated in utero at E14.5 with a plasmid encoding EGFP (green). DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. E Neurons from the brain sections in (D) were quantified and the total number of EGFP+ neurons in the Kcc2-/- sections is presented as a percentage of pooled Kcc2+/+ and Kcc2+/- values. Statistical significance was determined using one-tailed Student's t test, *P < 0.05. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos. F Number of EGFP+ neurons in Kcc2-/- embryos migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective pooled data from Kcc2+/+ and Kcc2+/- embryos. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Kcc2+/+ + Kcc2+/-. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.