EMBO/soda-nel huggingface dataset:
ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis (2022)
Figure 4

Figure 4. ENKD1 functions in the removal of CP110 from the mother centriole.

(A) A table summarizing the centrosomal localization of cilium-related proteins in ENKD1-depleted RPE1 cells.

(B, C) Immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment. Scale bar, 1 µm.

(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.

(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two-tailed t-test was performed. p < 0.01 (**), p < 0.001 (***).
ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis (2022)
Figure 5

Figure 5. ENKD1 regulates the CP110-CEP97 interaction.

(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.

(D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h.

(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.

(G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band.

(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two-tailed t-test was performed. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), ns, not significant.
ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis (2022)
Figure 6

Figure 6. ENKD1 competes with CEP97 in binding to CP110.

(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG.

(B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).

(E) Identification of the domains of ENKD1 mediating its interaction with CP110.

(F) Identification of the domains of CP110 mediating its interaction with ENKD1.

(G) Schematic diagrams showing the domains of ENKD1 and CP110 that mediate their interaction.

(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.

(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Nonparametric one-way ANOVA analysis was performed. p < 0.001 (***).

Supplemental information

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis

Ting Song, Yunfan Yang, Peng Zhou, Jie Ran, Liang Zhang, Xiaofan Wu, Wei Xie, Tao Zhong, Hongbin Liu, Min Liu, Dengwen Li, Huijie Zhao* & Jun Zhou*

*Corresponding authors:

H.Z. (huijiezhao@sdnu.edu.cn) or J.Z. (email: junzhou@sdnu.edu.cn).
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 1

Figure 1 - Identification of CDKL5 substrates using chemical genetics.

A Schematic of chemical genetic kinase substrate identification method. AS-CDKL5 utilizes benzyl- ATPγS to thiophosphorylate its substrates. Following trypsin digest, -SH containing peptides are captured on iodoacetyl agarose beads. Thiophosphate ester linked peptides are released by oxone induced hydrolysis while Cysteine containing peptides remain attached. Eluted phosphopeptides are analysed with liquid chromatography tandem mass spectrometry (LC MS/MS).

B, C CDKL51-352 autophosphorylation is shown. WT CDKL5 uses ATPγS, KD is inactive, F89A uses benzyl-ATPγS with reduced efficiency, F89A/ C152A mutant has rescued activity. Quantification of Thio-P signal is normalized for CDKL5 levels (HA) and KD background is subtracted. Fisher's LSD: n = 4 replicates. bn = benzyl.

D A list of novel CDKL5 substrate targets and phosphorylation sites identified. All phosphorylation sites are conserved in human. # MAP1S pS812 is identified based on sequence homology. Red = phosphorylated serine, bold = RPXS consensus sequence.

E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5. 50 ng (40 nM) AS-CDKL5 phosphorylates 150 ng (50 nM) MAP1S very rapidly (E, F). In 30 minutes of incubation, 150 ng MAP1S is phosphorylated by small amounts of CDKL5 (G, H). Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates.

I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutants ARHGEF2 S122A (I), EB2 S222A (J) and MAP1S S786/812A (K). Substrate levels are shown by coommassie staining underneath. MAP1S phosphorylation is quantified in (L). Dunnett's multiple comparison: n = 3 replicates.

Data information: Thio-P = anti-thiophosphate ester antibody, n.s. = not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001, error bars are SEM. In panel (K), non-relevant lanes have been removed for simplicity (see accompanying Source Data for full blot).
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 2

Figure 2 - MAP1S and EB2 are physiological substrates of CDKL5 in mammalian brain.

A, B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT (CDKL5 +/Y) and 5 CDKL5 KO (CDKL5 -/Y) mouse cortical lysates using phosphospecific antibodies. EB2 pS222 and MAP1S pS812 are dramatically reduced in KOs. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 5 animals per genotype.

C, D Developmental expression of CDKL5 and levels of its substrate phosphorylations in P4 - P50 cortex. CDKL5-dependent substrate phosphorylation is quantified by subtracting KO from WT levels for each age. n = 3 animals per age per genotype.

E, F Expression of CDKL5 and levels of substrate phosphorylation in P8 Nex-Cre conditional KO cortex. CDKL5 protein expression is almost completely lost. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 3 animals per genotype.

G CDKL5 WT and CDKL5 KO neurons in culture are co-stained with neuronal dendrite marker MAP2, EB2 and EB2 pS222. EB2 pS222 is lost in CDKL5 KO dendrites. Scale bar is 20 μm.

Data information: EB2 isoform 1/2 (37 kD) is quantified. **** p < 0.0001, error bars are SEM.
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 3

Figure 3 - Phosphorylation of EB2 Ser222 is suppressed by NMDAR activity.

A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates.

D, E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations. Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point. EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates.

Data information: * p < 0.05, ** p < 0.01, *** p < 0.001, error bars are SEM.
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 4

Figure 4 - Microtubule dynamics are reduced in CDKL5 KO dendrites via MAP1S.

A Representative kymographs of EB3-tdTomato in cortical neuron dendrites at DIV14.

B - D In CDKL5 KOs (CDKL5 -/Y), comet distance (B) and lifetime (C) is increased when compared to WT (CDKL5 +/Y). Comet velocity (D) is unchanged. Student's t-test: n = 16/615 and 15/381 neurons/comets in WT and KO, respectively.

E Representative kymographs of CDKL5 WT and KO neurons transfected with control shRNA and MAP1S shRNA.

F Comet lifetime is higher in control shRNA expressing KOs than WTs. MAP1S shRNA expression reduces comet lifetime in KO neurons. EB2 shRNA-mediated knockdown does not alter microtubule dynamics in WT or KO. Tukey's multiple comparison: n = 12-15 neurons, 388-741 comets per condition.

Data information: n.s. = not significant ** p < 0.01, **** p < 0.0001, error bars are SEM.
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 5

Figure 5 - TrkB trafficking is impaired in CDKL5 KO dendrites.

A, B Representative stills (A) and kymographs (B) of TrkB-RFP overexpressed in dendrites of WT and CDKL5 KO DIV14 cortical neurons. Scale bar is 5 μm.

C, D No significant change is observed in the ratio of dynamic vs stationary vesicles (C) or anterograde vs retrograde runs (D). S = stationary, D = dynamic, A = anterograde, R = retrograde. Vesicles with a net distance > 5 μm were considered dynamic.

E, F Both anterograde and retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons (E). Anterograde run lengths are significantly reduced in KO compared to WT neurons (F).

Data information: Student's t-test: n = 4 cells/121-138 runs per condition. n.s. = not significant * p < 0.05, error bars are SEM.
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 6

Figure 6 - MAP1S phosphorylation by CDKL5 impairs its microtubule binding.

A Schematic representation of MAP1S protein domains. Phosphorylation sites S786 and S812 (green Ps) are on the microtubule binding domain of MAP1S LC. Scissors indicate proteolytic cleavage.

B COS-7 cells expressing HA-MAP1S LC, Flag-CDKL5 WT / kinase dead (KD) is shown with endogenous α-tubulin staining. MAP1S does not localize to microtubules when phosphorylated by CDKL5. Scale bar is 20 μm.

C, D The percentage of cells where α-tubulin and MAP1S LC are colocalised (C) and the quantification of MAP1S LC and α-tubulin colocalisation by Pearson correlation coefficient (D). Tukey's multiple comparison: n = 22-24 cells per condition.

E, F Taxol-stabilised microtubule co-sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5. Quantification of soluble MAP1S in the supernatant (s/n) fraction is normalized to the MT-bound pellet fraction. 3:1 MT:MAP1S LC ratio used here. Tukey's multiple comparison: n = 3 technical repeats.

G HA-MAP1S LC is overexpressed and stained with anti-HA antibody in cultured cortical neurons (red). Numerous loops at the dendrites' tips are observed (arrowheads). Scale bar is 5 μm.

Data information: * p < 0.05, *** p < 0.001, **** p < 0.0001, error bars are SEM.
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics (2018)
Figure 7

Figure 7 - EB2 S223 phosphorylation is lost in CDD patient-derived neurons.

A, B Western blots for total EB2, pS223 and tubulin using protein lysates obtained from neurons derived from three patients with CDD and their related control. 1 = p.D135_F154del, male; 2 = p.R59X, male; 3 = p.R59X, female. Quantification of EB2 phosphorylation is normalized for total protein level. Student's t-test: n = 3 patients/controls. * p < 0.05, error bars are SEM.

C Representative images of human iPSC-derived neurons co-stained with the neuronal dendrite marker MAP2, EB2 and EB2 pS223. EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons. Enlarged images of the boxed areas are shown on the right panel. Scale bar is 20 μm.

Data information: human EB2 pS223 is mouse/rat EB2 pS222
Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients (2016)
Figure 1

Figure 1 - Chronic oxidative stress reduces total H2AX protein levels and prevents normal accumulation of γ-H2AX

A Up, Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD-/- fibroblasts (Left) or in wt and Nfe2l2-/- fibroblasts (Right). Two representative clones, with their corresponding controls, are shown per genotype. H2B was used as negative control. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). n ≥ 3 independent experiments.

B Up, Representative western blot from kidney whole cell extracts showing H2AX protein levels in age-matched wt and junD-/- mice. Mice are 2 months old (young) and 18 months old (old) mice, respectively. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 5 mice per age and per genotype.

C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype.

D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments.

F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, following 2 Gy of γ-irradiation (F) or after camptothecin (CPT) treatment (H) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.

G,I Up, Representative γ-H2AX immunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after γ-irradiation (2Gy, 45 min) (G) or after camptothecin (CPT) treatment for 1h (I).. Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.

For all panels, data are shown as means ± s.e.m. P-values are based on Student t-test (A,B,C,G,I). P-values show the difference between wt and junD-/- or between wt and Nfe2l2-/- fibroblasts across time points and are based on paired t-test (D,E,F,H). Actin is used as internal control for protein loading (A,B,C,D,E,F,H). NS stands for not significant and * stands for P-value = 0.01. Scale bars = 10μm. a.u. stands for arbitrary unit.
Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients (2016)
Figure 2

Figure 2 - junD-deficient fibroblasts exhibit cell cycle delay, decreased cell survival and increased DNA damage.

A Bar plot showing the doubling time of wt and junD-/- fibroblasts.

B Representative cell cycle distribution of asynchronous wt and junD-/- fibroblasts.

C Bar plots showing the percentage of wt and junD-/- fibroblasts in G1, S and G2 phases of the cell cycle, following serum stimulation for the indicated times (hours, h), after 48h serum starvation.

D Representative views of alkaline comet assays from wt and junD-/- fibroblasts either untreated or after 10Gy γ-irradiation for the indicated times (minutes, min).

E Alkaline comet assays from wt and junD-/- fibroblasts at basal state. Left, Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts. Right, Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments. At basal state, 16% of junD-/- cells exhibit tail moment higher than 5, while it is the case for only 3% of wt cells.

F Alkaline comet assays from wt and junD-/- fibroblasts after 10Gy γ-irradiation. Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts, following 10Gy γ-irradiation for the indicated times (minutes, min).

G Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments, following 10Gy γ-irradiation for the indicated times (minutes, min).

H Surviving fraction of wt and junD-/- fibroblasts 7 days after 2Gy or 5Gy γ-irradiation, as assessed by clonogenic assay. Surviving fraction is expressed as percentage (%) of number of colonies after irradiation normalized with respect to untreated conditions.

I Surviving fraction of wt and junD-/- fibroblasts either transfected with an empty vector (+ Ctl) or H2AX-expressing (+ H2AX) vector. Surviving fraction is expressed as percentage (%) of alive cells, 72 hours post-transfection.

For all panels, data are shown as means ± s.e.m. n = 3 independent experiments (A-C,H,I) and at least 100 nuclei per genotype per condition have been analysed for comet assays (E-G). P-values are based on Student’s t-test (A,C,E Left,F,H,I) and on Fisher’s exact test (E Right, G).
Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients (2016)
Figure 4

Figure 4 - H2AX staining is reduced both in stroma and epithelium of aggressive BC

A Up, Representative views of H2AX immunostaining from 117 BC patients of Luminal-A (LumA, N=33), HER2 (N=37) and Triple-Negative (TN, N=47) subtypes. Scale bars = 100 μm. Down, representative views at high magnification of H2AX immunostaining focused on the Fibroblasts (Left) or Epithelium (Right) cells. Scale bars = 25 μm.

B Scatter plots of H2AX histological score (Hscore = staining intensity (0 to 4) x % of positive cells quantified from H2AX immunostaining (as shown in A) in Fibroblasts (Left) or Epithelial (Right) cells from LumA, HER2 and TN breast tumours.

C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells (as shown in B), in HER2 (left) and TN (right) breast tumours.

D Representative views of smooth muscle actin (SMA) immunostaining from BC patients (N=29) of Luminal-A (LumA, N=10), HER2 (N=10) and TN (N=9) subtypes. Scale bars = 100 μm. Zoom views are focused on the fibroblast compartment. Scale bars = 25 μm.

E Scatter plots of SMA histological score (Hscore) evaluated from SMA immunostaining (as shown in D).

F Correlation plot between SMA and H2AX Hscores in stromal compartment.

G Scatter plot of the mitotic index, evaluated by pathologists, in LumA, HER2, and TN BC patients, as indicated.

H Absence of correlation between mitotic index and epithelial H2AX Hscore, in LumA HER2 and TN BC.

Data are shown as mean ± s.e.m. (B,E,G). P values are based on Welch’s t test (B) or Student’s t-test (E,G). Correlation coefficients σ and P-value are based on Spearman’s rank correlation test (C,F,H).