EMBO/soda-nel huggingface dataset:
Quaking 5 suppresses TGF-β-induced EMT and cell invasion in lung adenocarcinoma (2021)
Figure 1

Figure 1. Identification of metastasis-associated QKI in human LUAD.

(A) Four lung cancer data sets including TCGA_LUAD database, and GSE40791, GSE75037, GSE7670 from GEO database were intersected with human RBP database ATtRACT to identify the differentially expressed RBPs in LUAD. The Venn diagram depicts the overlap between 5 independent data sets.

(B) The upper panel showing qRT-PCR analysis of four identified LUAD-associated RBPs (ADARB1, CELF2, QKI and ZFP36) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues. The lower panel showing relative mRNA expression (T/N) of the four RBPs in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. LUAD tissues were classified into metastatic and non-metastatic tissues as described in Materials and Methods. T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression (T/N) of four RBPs. Data are shown as the mean ± SD. ns, not significant; **P < 0.01 and ***P < 0.001 by unpaired Student's t-test.

(C and D) Comparison of QKI mRNA and protein expression between normal lung tissues and LUAD tissues, which were stratified by clinical stages (Stages 1-4) in TCGA database. Data are shown as the boxplot. Central band: median; Upper and lower line of boxes: upper and lower quartile; Upper and lower line of whiskers: maximum and minimum. P < 0.001 compared to normal lung tissues by unpaired Student's t-test.

(E-G) Kaplan-Meier survival curves (http://kmplot.com/analysis/) of LC, LUAD and LUSC patients (n=1540, n=865 and n=675, respectively) with high or low expression levels of QKI. The log-rank test was used to analyze the difference between two groups.

(H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299. Data are shown as the mean ± SD of n = 3 technical replicates. ***P < 0.001 compared to BEAS-2B cells by unpaired Student's t-test.

(I) Western blot analysis of QKI-5 protein levels in BEAS-2B, A549 and H1299 cells. β-actin was used as an internal control.

(J and K) qRT-PCR analysis of QKI-5 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (J) and T/N mRNA expression of QKI-5 in 61 paired LUAD tissues (K). T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of QKI-5. In the violin plot, central dotted line represents median, upper and lower dotted line represents quartile (J). ***P< 0.001 by unpaired Student's t-test.

(L) Violin plots showing QKI-5 mRNA expression (T/N) in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile. *P< 0.05 by unpaired Student's t-test.
Quaking 5 suppresses TGF-β-induced EMT and cell invasion in lung adenocarcinoma (2021)
Figure 2

Figure 2. QKI-5 directly interacts with TGFβR1 mRNA by targeting TGFβR1 3' UTR in LUAD cells.

(A) The Venn diagram depicts the identification of 2511 QKI-targeted genes overlapping between three RBP-binding sites databases (POSTAR, EuRBPDB and doRiNA).

(B) KEGG enrichment analysis was performed by clusterProfiler in R language. Top 20 interested KEGG enrichment terms of 2511 QKI-targeted genes identified in Fig 2A were shown in left panel. A total of 22 genes were enriched in TGF-β signaling pathway, which were presented in right panel.

(C) Western blot analysis of QKI-5 expression in nuclear and cytoplasmic fractions extracted from A549 and H1299 cells. Lamin B and β-actin were used as internal controls in nuclear and cytoplasmic extracts, respectively.

(D) Anti-QKI-5 RIP was conducted in nuclear and cytoplasmic extracts from A549 cells and H1299 cells, and followed by RT-PCR and gel staining analyses to examine TGFβR1 mRNA enrichment. The input and anti-IgG antibody were used as positive and negative controls, respectively. GAPDH served as an internal RNA control in RT-PCR analysis.

(E) After RIP assay, endogenous TGFβR1 mRNA was enriched from A549 cells and H1299 cells and determined by qRT-PCR. Results were shown as the percentage of the enrichment to input. Data are shown as the mean ± SD of n = 3 technical replicates. **P < 0.01; ***P < 0.001 by unpaired Student's t-test.

(F) A conserved consensus QKI-5 response element (QRE) was presented.

(G) Schematic drawing shows two predicted QREs in TGFβR1 mRNA 3' UTR (upper panel). Boxed areas indicate several TGFβR1 3' UTR segments (positions, 2,058-2,557) containing wild-type (TGFβR1 3' UTR-WT) or mutants of QRE-1/2 (TGFβR1 3' UTR-Mut1, TGFβR1 3' UTR-Mut2, and TGFβR1 3' UTR-Mut1*2) (lower panel), which are prepared to subclone into pGL3-Luc reporter vector. Numbers represent the nucleotide position of TGFβR1 mRNA.

(H) Western blot analyses of QKI-5 expressed in A549 and H1299 cells stably overexpressing Flag-tagged QKI-5.

(I) Left panel: schematic drawing of the pGL3-TGFβR1 3' UTR luciferase reporter assay system. Middle and right panels: QKI-5-overexpressing and vector A549 and H1299 cells were transfected with pGL3-Luc reporter plasmids that were made by fusing the above-mentioned TGFβR1 3' UTR segments. Relative luciferase activities were determined as described in Materials and Methods and normalized to those in vector cells. Data are shown as the mean ± SD of n = 3 technical replicates. *P< 0.05, **P < 0.01 and ***P < 0.001 by unpaired Student's t-test.

(J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.
Quaking 5 suppresses TGF-β-induced EMT and cell invasion in lung adenocarcinoma (2021)
Figure 3

Figure 3. QKI-5 negatively regulates TGFβR1 and TGF-β/SMAD signaling in LUAD cells.

(A and B) qRT-PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI-5-overexpressing A549 and H1299 cells. Data are shown as the mean ± SD of n = 3 technical replicates. **P < 0.01 by unpaired Student's t-test.

(C and D) Expression of TGFβR1 mRNA and protein in QKI-5-silenced A549 and H1299 cells. **P < 0.01 and ***P < 0.001 by unpaired Student's t-test.

(E and F) The mRNA stability of TGFβR1 was examined in QKI-5-overexpressing/-silenced and control A549 and H1299 cells. Cells were treated with actinomycin D (Act-D, 10 μg/ml) for 0, 2, 4 or 6 h, and mRNA levels of TGFβR1 were determined by qRT-PCR. The quantity value of qRT-PCR at 0 h was set 1. *P < 0.05 and **P < 0.01 by unpaired Student's t-test.

(G and H) After being serum starved for 24 h, A549 and H1299 cells stably overexpressing QKI-5 were treated with or without TGF-β1 (5 or 10 ng/ml) for 24 h and subjected to western blot assays to determine the protein levels of QKI-5, TGFβR1, p-SMAD3 and SMAD3. β-actin was used as an internal control.

(I and J) QKI-5-silenced A549 and H1299 cells were treated as above. Then, western blot analysis was conducted to examine the protein levels of QKI-5, TGFβR1, p-SMAD3 and SMAD3.

(K) QKI-5-overexpressing A549 and H1299 cells were treated as above and subjected to qRT-PCR assays to detect the mRNA expression of downstream genes of the TGF-β/SMAD signaling, including PAI-1, Slug, Snail, E-cadherin, and/or N-cadherin. Data are shown as the mean ± SD of n = 3 technical replicates. *P < 0.05, **P < 0.01 and ***P < 0.001 by unpaired Student's t-test.
Quaking 5 suppresses TGF-β-induced EMT and cell invasion in lung adenocarcinoma (2021)
Figure 4

Figure 4. QKI-5 overexpression inhibits TGF-β-induced EMT and migration and invasion of LUAD cells.

(A) After serum-starvation for 24 h, A549 and H1299 cells overexpressing QKI-5 were treated with or without TGF-β1 (5 or 10 ng/ml ) for 24 h. Then, EMT markers N-cadherin and Snail expression were analyzed using western blot. β-actin served as an internal control.

(B) QKI-5-overexpressing A549 and H1299 cells and control cells were treated as above and incubated with a mouse anti-F-actin antibody, and then stained with a FITC-conjugated anti-mouse IgG (green, for F-actin). Cell nuclei were counterstained and visualized with DAPI (blue). Scale bar, 100 μm.

(C-F) Wound-healing migration assays were performed on QKI-5-overexpressing A549 and H1299 cells as well as control cells, which were treated as above. The wound healing was recorded (C and E) and quantitatively measured (D and F) at least six times. Data are shown as the mean ± SD of n = 6 technical replicates. **P < 0.01 and ***P < 0.001 by unpaired Student's t-test. Scale bar, 100 μm.

(G and H) In the presence or absence of TGF-β1, QKI-5-overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells. After 24 h, migrated cells were stained, photographed and counted in at least four random fields under a light microscope. Representative images (left) and the migrated cell numbers (right) were presented. Data are shown as the mean ± SD of n = 3 technical replicates. **P < 0.01 by unpaired Student's t-test. Scale bar, 100 μm.

(I and J) QKI-5-overexpressing A549 and H1299 cells were treated as above and allowed to invade through Matrigel-coated membrane in Transwells. Invasive cells were determined as described above. Scale bar, 100 μm. Data are shown as the mean ± SD of n = 3 technical replicates. **P < 0.01 by unpaired Student's t-test.
Quaking 5 suppresses TGF-β-induced EMT and cell invasion in lung adenocarcinoma (2021)
Figure 6

Figure 6. QKI-5 inhibits TGF-β-induced EMT and cell invasion via a TGFβR1-dependent manner.

(A) A549 and H1299 cells were co-transfected with QKI-5 and ORF-only of TGFβR1, and then subjected to western blot assays for detection of QKI-5 and TGFβR1 expression.

(B) After serum-starvation for 24 h, the above-treated A549 and H1299 cells were stimulated with or without TGF-β1 (5 or 10 ng/ml) for 24 h. The expression of p-SMAD3, SMAD3 and EMT markers (N-cadherin and Snail) was determined using western blot. β-actin was used as an internal control.

(C-F) In the presence or absence of TGF-β1, the migratory and invasive capabilities of above-treated A549 and H1299 cells were evaluated by Transwell assays as described in Fig 4G-J. Migrated and invasive cells were stained and counted under a light microscope. Data are shown as the mean ± SD of n = 3 technical replicates. **P < 0.01 and ***P < 0.001 by unpaired Student's t-test. Scale bar, 100 μm.

(G and H) qRT-PCR analysis of TGFβR1 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (G) and T/N mRNA expression of TGFβR1 in 61 paired LUAD tissues (H). T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of TGFβR1. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (G). ****P< 0.0001 by unpaired Student's t-test.

(I) Correlation between QKI-5 and TGFβR1 mRNA expression in 61 paired LUAD tissues. X and y axes represent the log10 transformed fold change of T/N expression ratios of QKI-5 and TGFβR1 mRNA, respectively. P< 0.0001 by Pearson's correlation test.

(J) Kaplan-Meier survival curves (http://kmplot.com/analysis/) of LUAD patients (n=865) with high or low expression levels of TGFβR1. The log-rank test was used to compare the difference between two groups.
Quaking 5 suppresses TGF-β-induced EMT and cell invasion in lung adenocarcinoma (2021)
Figure 7

Figure 7. KLF6 transcriptionally regulates QKI-5 gene in LUADs.

(A) Four databases used in Fig 1A were intersected with human TFs database JASPAR to identify differentially expressed TFs in LUAD. The Venn diagram depicts the overlap between 5 independent data sets.

(B) Listed are twenty-five LUAD-related differentially expressed TFs. TFBSTools was used to predict potential binding sites of each TF in QKI-5 promoter sequence (position -3000 to +200). TFBSTools minscore > 95%; TFs, transcription factors.

(C and D) qRT-PCR analyses of mRNA expression of ten TFs and QKI-5 in A549 cells transfected with siRNAs of TFs, which have potential binding sites in QKI-5 promoter. Data are shown as the mean ± SD of n = 3 technical replicates. ***P < 0.001 compared to si-NC by unpaired Student's t-test.

(E) Western blot analysis of QKI-5 protein levels in KLF6-silenced A549 cells.

(F) An overview of KLF6-ChIP-seq data (GSE96355) is illustrated using Integrative Genomics Viewer (IGV) software. An obvious peak was detected at position -100 to +100 in the QKI-5 promoter.

(G) Upper panel: computational algorithms using PROMO and JASPAR predicted that two putative KBEs were harbored in ΔQP region (position -400 to +200). QP, QKI-5 promoter; KBE, KLF6 binding element. Lower panel: anti-KLF6 ChIP was conducted in A549 cells and followed by PCR and gel-staining to confirm the presence of QKI-5 promoter sequence containing KBE1 (position -346 to -336) or KBE2 (position +21 to +31). The input and anti-IgG antibody were used as positive and negative controls, respectively.

(H) Left panel: schematic drawing shows the pGL3-ΔQP luciferase reporter assay system. Boxed areas indicate four ΔQP segments containing wild-type or mutants of KBE1/2 (Mutant 1, Mutant 2, and Mutant 1*2). Right panel: four different pGL3-ΔQP luciferase reporter constructs were co-transfected with si-NC or si-KLF6 into A549 cells, and their luciferase activities were determined as described in Materials and Methods. Data are shown as the mean ± SD of n = 3 technical replicates. *P < 0.05 compared to si-NC by unpaired Student's t-test.

(I and J) qRT-PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (I) and T/N mRNA expression of KLF6 in 61 paired LUAD tissues (J). T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of KLF6. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (I). ***P< 0.001 by unpaired Student's t-test.

(K) Correlation between KLF6 and QKI-5 mRNA expression in 61 paired LUAD tissues. X and y axes represent the log10 transformed fold change of T/N expression ratios of KLF6 and QKI-5 mRNA, respectively. P = 0.002 by Pearson's correlation test.

(L) A proposed work model: KLF6/QKI-5/TGFβR1 axis regulates TGF-β/SMAD signaling during TGF-β-induced EMT.

Expanded View Figure Legends
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 1

Figure 1. Abnormal neuronal migration in CAMDI-KO mice

(A) Abnormal distribution of Cux1-positive neurons in CAMDI-KO mice. Expression of Cux1 in the somatosensory cortex was compared between P2 wild-type (WT) and CAMDI-KO (KO) mice. Scale bar, 100 μm.

(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT = 788 cells, KO = 2,139 cells). *, p<0.05, **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe’s post-hoc test. Data are presented as mean ± SEM.

(C) Delay in neuronal migration by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Scale bar, 100 μm.

(D) Quantification of the number of EGFP-positive neurons. Note the abnormal distribution of neurons in lower cortical layers of CAMDI-KO mice. n = 3 for WT mice (546 cells), n = 4 for KO mice (680 cells). *, p<0.05, ***, p<0.001. Two-way ANOVA followed by Scheffe’s post-hoc test. Data are presented as mean ± SEM.

(E) Aberrant axonal projection and terminal arbors by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Representative pictures show an aberrant axonal projection of corticostriatal neurons in CAMDI-KO mice. Sections were stained with Hoechst 33258 and anti-EGFP antibody. Scale bar, 1 mm.

(F) Quantification of the striatal intensity. n = 3 mice/genotype. ***, p<0.001, One-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM.
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 2

Figure 2. Psychiatric behaviors in CAMDI-KO mice

(A, B) Representative traces from adult control (WT) and CAMDI-KO (KO) in open field test. Locomotor activity (A) and distance traveled (B) for 15 min. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni’s post hoc test (F(1, 13) = 5.53). Data are presented as mean ± SEM.

(C) Repetitive jumping behavior in open field test. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni’s post hoc test (F(1, 13) = 5.09). Data are presented as mean ± SEM.

(D) Three-chamber social interaction test. The time spent sniffing with stranger mouse. n = 19 for WT mice, n = 15 for KO mice. *, p<0.05, ***, p<0.001,Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 63)=5.79, main effect of chamber F(1, 63)=90.47, interaction F(1, 63)=5.29). Data are presented as mean ± SEM.

(E) Resident-intruder test (social sniffing). n = 7 for WT mice, n = 8 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni’s post hoc test (F(1, 13) = 5.15). Data are presented as mean ± SEM.

(F) Resident-intruder test (digging). n = 7 for WT mice, n = 8 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni’s post hoc test (F(1, 13) = 10.97). Data are presented as mean ± SEM.

(G) Marble burying test. n = 9 for WT mice, n = 25 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni’s post hoc test (F(1, 32) = 5.39). Data are presented as mean ± SEM.

(H) Forced swimming test. n = 10 for WT mice, n = 13 for KO mice. **, p<0.01, ***, p<0.001 Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 42)=84.30, main effect of session F(1, 42)=33.88, interaction F(1, 42)=4.92). Data are presented as mean ± SEM.

(I, J) Light-dark test. Time in light chamber (I) and Latency to light chamber (J). n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni’s post hoc test, ((I) F(1, 13) = 8.75, (J) F(1, 13)=9.73). Data are presented as mean ± SEM.

(K) Hot plate test.. n = 8 for WT mice, n = 12 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni’s post hoc test (F(1, 18) = 17.51). Data are presented as mean ± SEM.
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 3

Figure 3. CAMDI interacts with HDAC6 α-tubulin deacetylase

(A) Co-expressed FLAG-CAMDI and HDAC6-HA interacted with each other in SH-SY5Y cells. n=3 independent experiments.

(B) Endogenous interaction of CAMDI with HDAC6 in the mouse brain. Co-IP assay was performed with the indicated antibodies using E16 mouse brain. n=3 independent experiments.

(C, D) N-terminal region of CAMDI recognizes the second HDAC domain of HDAC6. SH-SY5Y cells were co-transfected with HDAC6-HA and each FLAG-CAMDI fragment (C) (fragment 1, 1–250; 2, 251–500; 3, 501-750; 4, 751-1000; 5, 1001-1451 amino acids) or co-transfected with FLAG-CAMDI and each HDAC6-HA fragment (D) (fragment 1 (DD1), 1–440; 2 (DD2), 441–840; 3, 841-1215 amino acids). Co-IP assay was performed with the indicated antibodies. n=3 independent experiments. **, p<0.01, ***, p<0.001, One-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM.

(E) Schematic illustration of interaction between CAMDI and HDAC6.
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 4

Figure 4. HDAC6 inhibition by CAMDI regulates Ac-Tubulin level at the basal bodies

(A) CAMDI and CAMDI Fragment 1 overexpression enhanced α-tubulin acetylation. HeLa cells overexpressing FLAG-CAMDI and FLAG-CAMDI frag.1 were immunostained with anti-FLAG or anti-Ac-tubulin antibodies. Ratio line-scans along the line (from x- to y-axis) from images. Scale bar, 10 μm.

(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (B) Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and Ac-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe’s post-hoc test. Data are presented as mean ± SEM.

(C) CAMDI N-terminal domain alone increased Ac-tubulin level. n=3 independent experiments. **, p<0.01, One-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM.

(D) Deletion of CAMDI N-terminal domain (1–250 amino acids) failed to inhibit HDAC6. n = 3 independent experiments. N.S., not significant. Two-way ANOVA followed by Scheffe’s post-hoc test. Data are presented as mean ± SEM.
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 5

Figure 5. HDAC6 inhibition by CAMDI regulates γ-tubulin localization to the centrosome

(A) Effect of HDAC6 over-expression or CAMDI co-expression on the level of acetylated α-tubulin in the centrosome. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-acetylated α-tubulin antibodies (red). Scale bar, 1 μm.

(B) Quantification of intensity of acetylated α-tubulin at centrosome. n = 25, 30, 30 cells. *, p<0.05, One-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM.

(C) Effects of HDAC6 overexpression or CAMDI co-expression on the centrosomal localization of γ-tubulin. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). DNA was stained with Hoechst 33258. Scale bar, 1 μm.

(D) Quantification of intensity of γ-tubulin at centrosome. n = 39, 16, 17 cells. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM.

(E) CAMDI stabilizes centrosomal γ-tubulin through HDAC6 inhibition. Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni’s post hoc test. Data are presented as mean ± SEM.

(F) Effects of CAMDI knockdown on the centrosomal localization of γ-tubulin in vivo.

Coronal sections through the somatosensory cortex of E17.5 were analyzed following in utero electroporation of EGFP and CAMDI-sh plasmid at E14.5. The sections were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). Representative images of EGFP at Cortical Plate neuron expressing CAMDI-sh stained for endogenous γ-tubulin and with Hoechst (blue). Z-stack number 6 indicates centrosome of EGFP-positive CAMDI-sh-expressing neuron. Z-stack number 19 indicates centrosome of a nearby non-electroporated neuron. Quantification of γ-tubulin intensity. EGFP (-) = 12 cells, EGFP (+) (CAMDI-sh) = 12 cells. *, p<0.05, One-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM. Scale bar, 5 μm.

(G) Reduced γ-tubulin in the centrosome fraction of CAMDI-KO brain. The centrosomal fraction was isolated from brain lysates of WT and CAMDI-KO mice and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni’s post hoc test. Data are presented as mean ± SEM.