EMBO/soda-nel huggingface dataset:
SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes (2016)
Figure 4

Figure 4. The predicted PUB domain in the N-terminus of SPATA2 interacts with the USP domain of CYLD.

A. Schematic representation of predicted domain structure of SPATA2. The figure shows domain structure of full-length SPATA2, and SPATA2 deletion mutants that were used for MS-based interaction analysis (see Figure 4B).

B. The predicted PUB domain in the N-terminus of SPATA2 interacts with CYLD, but not with LUBAC or other TNF-RSC components. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells that were stimulated with TNF-α for 15 minutes, and the enriched proteins were quantified using SILAC-based MS.

C. Validation of SPATA2 N-terminus interaction with CYLD. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells as described in Figure 5A, and association of CYLD was analyzed by immunoblotting. The lower panels show controls.

D. The USP domain of CYLD interacts with SPATA2. STREP-FLAG-CYLD and the indicated deletion mutants were purified from A549 cells using Step-Tactin sepharose and blotted with SPATA2 antibody. The lower panels show controls.

E. The C-terminus of SPATA2 interacts with HOIP. A549 cells were transiently transfected with GFP-tagged SPATA2, SPATA2 D200 and SPATA2 200*, and SPATA2 and the mutants were immunoprecipitated using GFP-trap beads. The immunoprecipitates were immunoblotted with HOIP, CYLD and GFP (HOIP) antibodies. The lower panels show input control, and the * shows unspecific band.
SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes (2016)
Figure 5

Figure 5. SPATA2 is necessary for the recruitment of CYLD to the TNF-RSC.

A. Strategy for identification SPATA2-dependent protein association with the TNF-RSC. SPATA2 was knocked down using RNAi, cells were stimulated with FLAG-TNF-α, and the TNF-RSC was enriched from control and SPATA2 knockdown cells as in Figure 3A. The enriched proteins were quantified using SILAC-based MS.

B. Effect of SPATA2 knockdown on association of proteins with TNF-RSC. The list shows SILAC ratios of TNF-RSC-associated proteins in control cells and SPATA2 knockdown cells. The proteins that showed reduced association with the TNF-RSC after SPATA2 knockdown are highlighted red.

C. Validation of SPATA2-dependent association of CYLD with the TNF-RSC. The TNF-RSC was purified under the indicated conditions and association of SPATA2, CYLD, RIPK1 and HOIP with the TNF-RSC was analyzed by immunoblotting. The input controls show expression of the analyzed proteins in cell lysates. The asterisk denotes unspecific bands.
SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes (2016)
Figure 6

Figure 6. SPATA2 is involved in TNF-α-induced NF-B activation and necroptosis.

A. SPATA2 regulates TNF-α-induced NF-κB activation. HEK293T cells were transfected with the SPATA2 siRNA (SMARTpool or with to individual siRNA) or a non-targeting control siRNA, and NF-B activation was monitored with luciferase-based NF-B reporter assays. Error bars specify the standard error of the mean (SEM) of three independent experiments. p** < 0.05 (two-tailed, unpaired Student´s t-test).

B. Knockdown of SPATA2 enhances expression of the NF-κB target genes. HEK293T cells were transfected with SPATA2 siRNA or a non-targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF-α for 0, 3 and 6 hours with qPCR.

C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test).

D. Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF-α-induced necroptosis. TNF-α-dependent necroptosis was induced in L929 as described in Figure 6C. Phosphorylation of MLKL was analyzed using S358 phospho-specific antibody.

E. Model for SPATA2 function in TNF-α signaling. SPATA2 interacts with CYLD and HOIP under steady-state conditions, and TNF-α-stimulation results in their recruitment to the TNF-RSC. SPATA2-dependent recruitment of CYLD impacts activation of TNF-α-induced NK-B signaling and necroptosis.
ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri (2018)
Figure 2

Figure 2: βHBP fails to induce rapid oligomerization of TIFA

A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cells (Hoechst in blue, IL-8 in red).

B) Quantification of IL-8 as shown in (A). Data represent the mean +/- SD of 3 independent experiments.

C) TIFA oligomerization at 6 h in TIFA-GFP-expressing HeLa cells treated as in (A).

D) Quantification of TIFA oligomerization as shown in (C). Data correspond to the mean +/- SD of 3 independent experiments.

E) TIFA oligomerization at 30 min in cells treated as in (A). Data correspond to the mean +/- SD of 4 independent experiments.

F) Kinetics of TIFA-GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S. flexneri lysate. Data correspond to the mean +/- SEM of 4 independent experiments.

Data information: Statistical significance was assessed using one-way ANOVA (B,D,E) or two-way ANOVA (F) followed by Tukey's multiple comparisons test *P<0.05, **P<0.01, ****P<0.0001, not significant (ns). (A,C): Scale bar, 20 μm.
ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri (2018)
Figure 3

Figure 3: ADP-heptose is a newly identified bacterial PAMP

A) LPS biosynthesis pathway. HldA and HldC from N. meningitidis are in brackets.

B) TIFA-GFP oligomerization at 30 min in HeLa cells treated with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC).

C) TIFA-GFP oligomerization at 6 h in cells treated as in (B).

D) IL-8 production at 6 h in cells were treated as in (C).

E) TIFA-GFP oligomerization in cells treated for 30 min with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations.

F) IL-8 production after 6 h in cells treated as in (E).

Data information: In (B-F), data are presented as mean +/- SD of 3 independent experiments. In (B-D), data were normalized to wt lysate treatment. Statistical significance was assessed using one-way ANOVA followed by Tukey's multiple comparisons test **P<0.01, ***P<0.001, ****P<0.0001, not significant (ns).
ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri (2018)
Figure 4

Figure 4: ADP-heptose sensing induces inflammation in S. flexneri infection

A) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed-expressing wt or indicated mutants of S. flexneri (MOI 50). pHldA/C means plasmid-encoded HldA and HldC. Fluorescence intensity was adjusted between strains to optimize visualization of bacteria. Scale bar, 20 μm.

B) Infectivity of indicated S. flexneri strains.

C) TIFA-GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway (MOI 10).

D) TIFA-GFP oligomerization after 4 h of infection with the S. flexneri mutants shown in (C).

E) IL-8 production 4 h post infection (MOI 5).

Data information: In (B-E), data are presented as mean +/- SD of 3 independent experiments. For comparison between mock and each infected condition (C-E) or ∆hldE and double-complemented mutant (B), statistical significance was assessed using one-way ANOVA followed by Tukey's multiple comparisons test **P<0.01, ***P<0.001, ****P<0.0001, not significant (ns).
ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri (2018)
Figure 5

Figure 5: ADP-heptose sensing is ALPK1-dependent

A) ADP-heptose-induced TIFA-GFP oligomerization depends on ALPK1. After siRNA transfection, HeLa cells were transfected with empty pCMV or pCMV-ALPK1 as indicated. They were then infected with wt S. flexneri (MOI 10) or treated with digitonin and ADP-heptose (10-5 M) for 30 min. As control, cells were left untreated (mock) or treated with digitonin.

B) ADP-heptose-induced cytokine secretion is ALPK1-dependent. HeLa cells were transfected with control or ALPK1-targeting siRNAs and infected with wt S. flexneri (MOI 2) or treated with ADP-heptose (10-5 M). As control, cells were left untreated (mock) or treated with digitonin. Cytokines were measured in cell culture supernatants after 8 h.

Data information: In (A-B), data correspond to the mean +/- SD of 3 independent experiments. Statistical significance was assessed using two-way ANOVA followed by Tukey's multiple comparisons test ***P<0.001, ****P<0.0001.

EXPANDED VIEW FIGURE LEGENDS
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 1

Figure 1. M. catarrhalis infection activates the non-canonical NLRP3 inflammasome and induces pyroptosis.

A. Volcano plot profiling of gene expression changes in WT BMDMs infected with M. catarrhalis (Ne11, MOI 100) for 8 h relative to untreated WT BMDMs. Upregulated differentially expressed genes (DEGs) are represented by red dots (p-value <0.01, counts per million >1 and fold-change >2) within the grey box.

B. Enrichment and similarity analysis of gene ontology terms associated with the significantly upregulated DEGs depicted in A.

C. Multiplex ELISA of secreted inflammatory cytokines (right) and associated gene expression by RNA-sequencing (left) of WT BMDMs infected with M. catarrhalis (Ne11) as in A.

D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11), gasdermin D (GSDMD) and GAPDH (loading control) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100).

E. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in
D.

F. Brightfield microscopy analysis of WT and mutant BMDMs after treatment as in D.

G. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability after infection with M. catarrhalis as in D, or transfection with LPS.

H. Scanning electron microscopy of WT BMDMs left untreated or assessed after infection as in D.

I. Transmission electron microscopy of WT BMDMs left untreated or assessed after infection as in D.

Data information: Arrowheads indicate dead cells. Each symbol represents an independent biological replicate (E). **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E). Data are from one experiment representative of two (H and I) or three independent experiments (D and F) or pooled from two (G) or three independent experiments (A-C, E; mean and s.e.m. in E and G). Scale bars, 20 µm (F), 2 µm (H and I).
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 2

Figure 2. M. catarrhalis infection and secreted OMVs deliver LOS to facilitate inflammasome activation.

A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs left untreated (Med.) or 5 h after transfection with 5 µg of LOS from M. catarrhalis (O35E) or 5 µg of LPS from E. coli.

B. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in A.

C. Immunoblot analysis of Casp-1, Casp-11 and GSDMD in WT, Nlrp3-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (O35E and ∆lpxA, MOI 100).

D. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in C.

E. Immunoblot analysis of Casp-1, Casp-11 and GSDMD in WT, Nlrp3-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after incubation with 10 µg of OMVs purified from M. catarrhalis (O35E and ∆lpxa).

F. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in
E.

G. Silver-stained SDS-PAGE of LOS purified from M. catarrhalis (O35E and ∆lpxa).

H, I. Transmission electron microscopy of negative stained OMVs purified from M. catarrhalis (O35E and ∆lpxa).

Data information: Each symbol represents an independent biological replicate (B, D, F). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B, D, F)). Data are from one experiment representative of two (G-I) or three independent experiments (A, C, E) or are pooled from three independent experiments (B, D, F; mean and s.e.m. in B, D, F). Scale bars, 100 nm (H and I).
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 3

Figure 3. IFN-inducible proteins are essential for inflammasome activation during M. catarrhalis infection.

A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT and Ifnar1-/-BMDMs left untreated (Med.) or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 50).

B. Release of IL-1β, IL-18, LDH and TNF from BMDMs after treatment as in A.

C. Volcano plot profiling of gene expression changes in Ifnar1-/- BMDMs, relative to WT BMDMs, infected with M. catarrhalis (Ne11, MOI 100) for 8 h. Differentially expressed genes (DEGs) are represented by red dots. Downregulated DEGs are shown in the grey box.

D. Gene ontology analysis of significantly downregulated DEGs (n=448) as depicted in
C.

E. Functional ontology analysis of the downregulated DEGs present within the top 5 biological processes depicted in C.

F. Transcript abundance for genes encoding GTPases in WT and Ifnar1-/- BMDMs infected with M. catarrhalis as in C. Transcript abundance is provided in counts per million (CPM).

G. qRT-PCR analysis of genes encoding the family of guanylate binding proteins (GBP1-11) in WT and Ifnar1-/- BMDMs left untreated (Med.) or following treated as in C.

Data information: Each symbol represents an independent biological replicate (B and G). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B and G)). Data are from one experiment representative of three independent experiments (A) or are pooled from three (B-F) or four (G) independent experiments (mean and s.e.m. in B and G). ND, not detected.
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 4

Figure 4. GBPs target intracellular M. catarrhalis, promote inflammasome activation and mediate bacterial clearance

A. Confocal microscopy analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20).

B. Quantitation of GBP1-, GBP2-, GBP3-, GBP5- and GBP7-postive M. catarrhalis in WT BMDMs as treated in A.

C. Confocal microscopy analysis of FLAG-OVA, FLAG-GBP1, FLAG-GBP2, FLAG-GBP3, FLAG-GBP5, FLAG-GBP7 (red) and M. catarrhalis (green) in LA-4 cells left untreated (Med.), or 16 h after infection with M. catarrhalis (Ne11, MOI 20) primed with IFN-γ (100 U/mL).

D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbpchr3-KO and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS from E. coli.

E. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in D.

F. Recovery of M. catarrhalis (as colony-forming units (CFU) from primary WT, Gbpchr3-KO and Ifnar1-/- lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100).

G. Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbpchr3-KO and Ifnar1-/- BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Each symbol represents an independent experiment (E and F).

Data information: Arrowheads indicate bacteria colocalised with GBPs. Each symbol represents an independent biological replicate (E and F). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E-G). To quantify the bacterial number in a field of view, we used the single green channel (anti-M. catarrhalis staining), where a single bacterium is defined as an intact circle. To quantify the proportion of GBP-positive bacteria in a field of view, we used the single red channel (anti-GBP staining) and compared this to the green channel (anti-M. catarrhalis staining). The number of GBP-positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP-positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP-positive bacteria per cell using confocal microscopy (B) or the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) or three independent experiments (A, C, D) or are pooled from two (B) or three independent experiments (E, F; mean and s.e.m. in B, E-G). Scale bars, 5 µm (A, C).
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 5

Figure 5. GBP1, 2, 3 and 5 are required while GBP4, 7, 8, 9 and 11 are dispensable for M. catarrhalis-induced inflammasome activation.

A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbp1-/-, Gbp2-/-, Gbp3-/-, Gbp5-/-, Gbp7-/-, Gbp4/8/9-/-, Gbp11­­-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS from E. coli.

B. Release of IL-1β, IL-18, LDH, TNF, IL-6 and KC from BMDMs after treatment as in A.

C. Brightfield microscopy analysis of WT and mutant BMDMs after treatment as in A.

Data information: Arrowheads indicate dead cells. Each symbol represents an independent biological replicate (B). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B)). Data are from one experiment, representative of three independent experiments (A and C) or are pooled from three independent experiments (B; mean and s.e.m. in B). Scale bars, 20 µm (C).
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 6

Figure 6. GBP2 targets and mediates killing of M. catarrhalis.

A. Correlative light electron microscopy (CLEM) of HEK293T cells overexpressing eGFP-GBP2 left untreated (Med.) or infected with M. catarrhalis (Ne11, MOI 5).

B. Quantification of OD600 of M. catarrhalis in BHI media over 6 h in the presence of solvent control (Sol. Ctrl.), 80 µg/mL recombinant GBP2 or 50 µg/mL kanamycin.

C. Quantification of ATP from M. catarrhalis in BHI media over 6 h in the presence of Sol. Ctrl., 30 µg/mL recombinant GBP2 or 50 µg/mL kanamycin.

D. Viability of M. catarrhalis (as a percentage of CFU in relation to Sol. Ctrl.) assessed 6 h after incubation with recombinant GBP2 at 3, 30 or 300 µg/mL.

E. Quantitation of DiOC2(3) stained M. catarrhalis with fluorescence emission shift from green to red (as a percentage relative to Sol. Ctrl.) assessed 6 h after incubation with recombinant GBP2 at 50 µg/mL or 5 µM CCP for 30 min.

F. Flow cytometry plots showing the gating strategy for analysis of fluorescence emission by DiOC2(3) stained bacteria.

G. Uptake of NPN in M. catarrhalis (as a percentage relative to 20 µg/mL polymyxin B treatment) assessed 15 min after incubation with Sol. Ctrl. or recombinant GBP2 at 40 µg/mL.

H. Flow cytometric quantitation of SYTOX-stained M. catarrhalis treated with 100 µg/mL of recombinant GBP2 for 6 h.

I. Transmission electron microscopy (TEM) analysis of negative stained M. catarrhalis 6 h after treatment with Sol. Ctrl. or 100 µg/mL of recombinant GBP2.

J. Scanning electron microscopy (SEM) analysis of the morphology of M. catarrhalis 6 h after treatment as in I.

Data information: White arrowheads indicate bacteria colocalised with GBP2. Yellow arrowheads indicate bacteria without GBP2 colocalisation. ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B and D) or two-tailed t-test (E, G, H)). Data are from one experiment representative of two (A) or three (B, D, G, I, J) independent experiments or are pooled from two (C) or three (E, H) independent experiments (mean and s.e.m. in B-E, G, H). Scale bars, 1 µm (A), 0.5 µm (I and J). Each independent experiment consists of two (C) or three (B, D, G) technical replicates.
Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes (2023)
Figure 7

Figure 7. GBP1, GBP2, GBP3 and GBP5 control bacterial numbers and inflammasome activation during M. catarrhalis infection in mice.

A, B. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Casp11-/- mice (n=10) after M. catarrhalis infection.

C, D. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Nlrp3-/- mice (n=11) after M. catarrhalis infection.

E, F. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp1-/- mice (n=9) after M. catarrhalis infection.

G, H. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp2-/- mice (n=8) after M. catarrhalis infection.

I, J. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp3-/- mice (n=8) after M. catarrhalis infection.

K, L. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=5) and Gbp5-/- mice (n=8) after M. catarrhalis infection.

Data information: A mix of male and female mice 6-8 weeks old were used in each experiment and were injected i.p. with 2×107 CFU of M. catarrhalis and analysed after 6 h. Each symbol represents an individual mouse (A-L). Each panel represents data from a single experiment (mean and s.e.m. in A-L). Each experiment was performed at least two times. * P<0.05; ** P<0.01; *** P<0.001 (two-tailed t-test (A-L)).

EXPANDED VIEW FIGURES
Ttyh1 regulates embryonic neural stem cell properties by enhancing the Notch signaling pathway (2018)
Figure 1

Figure 1. Ttyh1 enhances embryonic neural stem cell properties.

A-D Neurosphere (A, C) and XTT (B, D) assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1 or short hairpin RNA specific to Ttyh1 (shTtyh1) (n = 4 for A and C, n = 3 for B and D).

E Ttyh1-expressing cells in E15.5 embryonic brains that were intraventricularly electroporated at E13.5 were immunostained using an anti-GFP primary (reporter gene) and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.

F Quantification of (E) (n = 4).

G Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm.

H Quantification of (G) (n = 3).

I Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm.

J Quantification of (I) (n = 3).

Data information: Error bars represent SD. Student's t-test was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.