Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker

• ICC/IF

AbReview21462****

Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

ab27074 staining gamma tubulin in the Human HEK293T cells by ICC. Cells were fixed with paraformaldehyde, permeabilized with methanol (-20°C 20 min) and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/500 or 1/1000 in blocking solution) for 45 minutes. A Texas Red^®-conjugated Goat anti-mouse polyclonal (1/500) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Overlay histogram showing HeLa cells stained with ab27074 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab27074, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Separation of MCF-7 cells stained using ab27074 (concentration in sample 9 μg/ml, GAM APC, red-filled) from MCF-7 cells unstained by primary antibody (GAM APC, black-dashed) in flow cytometry analysis (intracellular staining).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry staining of P19X1 mouse embryonal carcinoma cell line using anti-gamma-tubulin (TU-30) (detection by secondary antibody Goat anti-mouse Cy3). Nuclei were stained with DAPI (blue).

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Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemisrty analysis of murine fibroblasts labeling gamma Tubulin with ab27074 at 1 ug/ml (red). Nuclei were stained with DAPI (blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

ab27074 staining gamma tubulin (red) in rat prostate carcinoma cells by ICC. The cells were also transfected with beta-tubulin GFP which stained green.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemisrty analysis of 3T3 mouse fibroblasts in metaphase (A), anaphase (B) and telophase (C), labeling gamma Tubulin with ab27074 at 1 ug/ml (red). Cells were costained for alpha-tubulin (green). Nuclei were stained with DAPI (blue).

• WB

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Western blot - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Western blotting analysis of gamma-tubulin using ab27074 on lysates of various cell lines under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2 μg/ml of ab27074 followed by IRDye800-conjugated anti-mouse secondary antibody. A specific band was detected for gamma-tubulin at approximately 46 kDa.

All lanes:

Western blot - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (ab27074)

Predicted band size: 51 kDa

Observed band size: 46 kDa

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

CEP44 is a centriolar protein necessary for the CCC mechanism.a IF of cycling RPE1 cells showing that CEP44 binds to the new dCs during G2. While S phase cells were detected by EdU stain, G1 and G2 cells were discerned by the lack of EdU stain and the number of centrin1 signals (centrioles). b IF of cells after 72 h of depletion. In the siCEP44 sample (lower panel) G1 cells contained less centrosomes as judged by the number of γ-tubulin foci (Cenp-F in Supplementary Fig. 1b). c Quantification of b. 80.4 ± 5.0% of G1 cells contained <2 centrosomes. d IF of 60 h siRNA treated RPE1 cells in G1. While the G1 control cells contained 2 defined PCM foci (γ-tubulin) accompanied by equal number of CEP44 foci, in the siCEP44 sample the loss of CEP44 correlated with inefficient PCM (γ-tubulin) recruitment to only one (bottom panel) or both centrosomes (middle panel) (Cenp-F, Supplementary Fig. 1g). e Quantification of CEP44 loss in d. 65.1 ± 7.3% of G1 cells contained <2 CEP44 foci. f Quantification of γ-tubulin defined foci in d. 60.7 ± 4.2% of G1 cells contained <2 defined γ-tubulin signals. g, h G2 CEP44-depleted cells after 60 h of CEP44 depletion showed a mild centriole duplication defect as judged by the counting of CEP97 foci (<4). g Quantification of h. 30.3 ± 4.1% of CEP44-depleted cells contained <4 centrioles. i MT regrowth assay in RPE1 C-Nap1 KO cells. The non-converted daughter centrosome without CEP164 staining regrew lower numbers of MTs (5.1 ± 2.7 MTs/centrosome) than the siControl daughter centrosomes (10.3 ± 3.0 MTs/centrosome) upon cold treatment and MT regrowth. j Quantification of i. k Loss of CEP44 leads to misalignment of mitotic spindles (>65%) generating either bipolar asymmetric spindles (51.3 ± 4.7%) or mono-/multipolar ones (14.3 ± 4.0). l Quantification of k. (a, b, d, h, i, k, scale bars : 10 µm, magnification scale bars : 1 µm; c, e, f, g, j and l data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file).

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

Structural integrity of the centrioles ensures their conversion to centrosomes.a–c EM images of turned and tilted sections of proximal region of daughter centrosomes in G1. The two values on the top of the image give the turning and tilting degree of the sample, respectively. Red arrowheads indicate open structural defects (A–C linker absent), while yellow ones highlight defects of triplets. The side table (right) shows quantification of the number of cells showing these defects. While in a G1 siControl cells had no structural defects, in b siTUBD1 and c siTUBE1 G1 dCs showed defects in triplet formation (yellow arrow). b siTUBD1 G1 cell with defect in the A–C linker formation on top of the triplet formation of dCs and lack of triplet MTs (red arrow). d Depletion of TUBD1 and TUBE1 proteins also generated reduced glutamylation of the centrioles as judged by GT335 staining (36.7 ± 2.2% of G1 cells, Cenp-F in Supplementary Fig. 12a) with <2 GT335 foci in siTUBE1 sample; 23.5 ± 2.0% for siTUBD1). e Quantification of d. f In absence TUBE1 or TUBD1 many centrioles in G1 cells (Cenp-F in Supplementary Fig. 12b) did not efficiently acquire PCM (<2 γ-tubulin defined foci) to convert to centrosomes. g Quantification of f. 33.1 ± 1.3% of cells with <2 γ-tubulin foci in G1 for siTUBE1; 26.2 ± 4.1% for siTUBD1. (a–c, scale bars : 100 nm; d, f scale bars : 10 µm, magnification scale bars : 1 µm; e, g data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file). h Model of the dependency of CCC mechanism on the correct centriolar wall maturation. See Discussion for description.

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

CEP44 localises to the centriole lumen via its MT-binding affinity.a–c 2D-SIM images of G1 centrioles (α-tubulin) and corresponding normalised intensity profiles of centrosomes positioned perpendicularly to the imaging plane (white arrows). a CEP44 localised in the centriole lumen as POC1B (b). c CEP295 decorates the outer centriolar wall. d 2D-SIM of centrosomes with duplicated centriole pairs co-stained withα-tubulin, CEP44 and POC1B. e Intensity profiles of dC cross-section. CEP44 (left) and POC1B (right) reside in the dC lumen. f (left) 2D-SIM of centrosomes with duplicated centriole pairs co-stained withα-tubulin and CEP295. f (right) Intensity profiles of dC cross-section. g CEP44 immuno-gold labelling in purified centrosomes (left). Red arrows indicate 10 nm gold particles. g (right) Distance of the gold particles from A-, B- and C-tubule of the same triplet respectively 21.9 ± 3.1 nm, 32.0 ± 4.7 nm, 43.1 ± 7.5 nm (all cases, n = 23 particles, data present mean ± s.d.). h Binding assay of recombinant CEP44-Flag and h5- mutant purified from E.coli to MTs. GST-Flag was used as control. Proteins were incubated with soluble polymerised tubulin. MTs with bound proteins were sedimented by centrifugation. The supernatant (S) and pellet (P) were analysed by IB forα-tubulin and Flag. Supplementary Fig. 8f shows Coomassie blue stain gel of purified proteins. i Schematic representation of CEP44 domain organisation. (Bottom) Comparison of CEP44 domain predicted secondary structure organisation with the MT-binding domain of EB1 and EB3 proteins. j The h5- and the NT-fragment could not rescue the CCC defect vs. CEP44-Flag. Constructs were mildly expressed by the addition of 2 ng/ml doxycycline. k Quantification of j and Supplementary Fig. 9c. While the CT-Flag was unable to rescue the loss of PCM (63.9 ± 3.2% of cells with <2 γ-tubulin foci) and the NT only partially (32.6 ± 3.8%), the h5- mutant generated a CCC defect even in the siControl (27.6 ± 2.3%) and a stronger CCC phenotype in the siCEP44 (76.1 ± 2.6%). Data presented as mean ± s.d., all statistics derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments. (a, b, c, d, f, scale bars : 1 µm; g, scale bar : 100 nm; j, scale bar 10 µm, magnification scale bar : 1 µm). (a–c, e–g, k) Source data are provided as a Source Data file.

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

The CEP44-POC1B complex is needed to convert centrioles to centrosomes.a Anti-Flag IP using CEP44-Flag from RPE1 cells was analysed for POC1B, POC1A and CEP295 by immuno-blotting (IB). GAPDH was used as input control. b Coomassie Blue stained gel of in vitro binding between purified, recombinant CEP44-Flag and purified, recombinant POC1B-HA. See Supplementary Fig. 5b for IB and 5c for Coomassie Blue stained gels of purified proteins used in the experiment. c Schematic representation of CEP44 protein sequence identity in vertebrata (referred to Supplementary Fig. 5a). d CEP44-Flag constructs that were designed based on c and used in e. e IB of input and eluted samples from RPE1 IPs using CEP44-Flag constructs as outlined in d. The CEP44-Flag IPs were tested for the presence of POC1B. GAPDH was used as loading control for the input. f, g 39.2 ± 2.8% of G1 cells in which POC1B was depleted show <2 γ-tubulin defined foci (Cenp-F in Supplementary Fig. 5g). h–j Loss of either CEP44 or POC1B in response to siRNAs depletion by one of them. h, i CEP44 loss upon CEP44 siRNA has a similar impact on POC1B loss from dCs. i Quantification of h. h, j CEP44 delocalisation was less severe than POC1B loss upon POC1B siRNA. j Quantification of h. Upon siPOC1B depletion, CEP44 delocalised (18.9 ± 4.3% of G1 cells) less sever than POC1B (45.5 ± 2.3%). k Schematic representation of the functional interdependency between the conversion molecules in the CCC mechanism. (f, h, scale bars : 10 µm, magnification scale bars : 1 µm; g, i, j data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file).

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CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

CEP44 is a centriolar protein necessary for the CCC mechanism.a IF of cycling RPE1 cells showing that CEP44 binds to the new dCs during G2. While S phase cells were detected by EdU stain, G1 and G2 cells were discerned by the lack of EdU stain and the number of centrin1 signals (centrioles). b IF of cells after 72 h of depletion. In the siCEP44 sample (lower panel) G1 cells contained less centrosomes as judged by the number of γ-tubulin foci (Cenp-F in Supplementary Fig. 1b). c Quantification of b. 80.4 ± 5.0% of G1 cells contained <2 centrosomes. d IF of 60 h siRNA treated RPE1 cells in G1. While the G1 control cells contained 2 defined PCM foci (γ-tubulin) accompanied by equal number of CEP44 foci, in the siCEP44 sample the loss of CEP44 correlated with inefficient PCM (γ-tubulin) recruitment to only one (bottom panel) or both centrosomes (middle panel) (Cenp-F, Supplementary Fig. 1g). e Quantification of CEP44 loss in d. 65.1 ± 7.3% of G1 cells contained <2 CEP44 foci. f Quantification of γ-tubulin defined foci in d. 60.7 ± 4.2% of G1 cells contained <2 defined γ-tubulin signals. g, h G2 CEP44-depleted cells after 60 h of CEP44 depletion showed a mild centriole duplication defect as judged by the counting of CEP97 foci (<4). g Quantification of h. 30.3 ± 4.1% of CEP44-depleted cells contained <4 centrioles. i MT regrowth assay in RPE1 C-Nap1 KO cells. The non-converted daughter centrosome without CEP164 staining regrew lower numbers of MTs (5.1 ± 2.7 MTs/centrosome) than the siControl daughter centrosomes (10.3 ± 3.0 MTs/centrosome) upon cold treatment and MT regrowth. j Quantification of i. k Loss of CEP44 leads to misalignment of mitotic spindles (>65%) generating either bipolar asymmetric spindles (51.3 ± 4.7%) or mono-/multipolar ones (14.3 ± 4.0). l Quantification of k. (a, b, d, h, i, k, scale bars : 10 µm, magnification scale bars : 1 µm; c, e, f, g, j and l data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file).

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CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

CEP44 is a centriolar protein necessary for the CCC mechanism.a IF of cycling RPE1 cells showing that CEP44 binds to the new dCs during G2. While S phase cells were detected by EdU stain, G1 and G2 cells were discerned by the lack of EdU stain and the number of centrin1 signals (centrioles). b IF of cells after 72 h of depletion. In the siCEP44 sample (lower panel) G1 cells contained less centrosomes as judged by the number of γ-tubulin foci (Cenp-F in Supplementary Fig. 1b). c Quantification of b. 80.4 ± 5.0% of G1 cells contained <2 centrosomes. d IF of 60 h siRNA treated RPE1 cells in G1. While the G1 control cells contained 2 defined PCM foci (γ-tubulin) accompanied by equal number of CEP44 foci, in the siCEP44 sample the loss of CEP44 correlated with inefficient PCM (γ-tubulin) recruitment to only one (bottom panel) or both centrosomes (middle panel) (Cenp-F, Supplementary Fig. 1g). e Quantification of CEP44 loss in d. 65.1 ± 7.3% of G1 cells contained <2 CEP44 foci. f Quantification of γ-tubulin defined foci in d. 60.7 ± 4.2% of G1 cells contained <2 defined γ-tubulin signals. g, h G2 CEP44-depleted cells after 60 h of CEP44 depletion showed a mild centriole duplication defect as judged by the counting of CEP97 foci (<4). g Quantification of h. 30.3 ± 4.1% of CEP44-depleted cells contained <4 centrioles. i MT regrowth assay in RPE1 C-Nap1 KO cells. The non-converted daughter centrosome without CEP164 staining regrew lower numbers of MTs (5.1 ± 2.7 MTs/centrosome) than the siControl daughter centrosomes (10.3 ± 3.0 MTs/centrosome) upon cold treatment and MT regrowth. j Quantification of i. k Loss of CEP44 leads to misalignment of mitotic spindles (>65%) generating either bipolar asymmetric spindles (51.3 ± 4.7%) or mono-/multipolar ones (14.3 ± 4.0). l Quantification of k. (a, b, d, h, i, k, scale bars : 10 µm, magnification scale bars : 1 µm; c, e, f, g, j and l data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file).

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Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

CEP44 is a centriolar protein necessary for the CCC mechanism.a IF of cycling RPE1 cells showing that CEP44 binds to the new dCs during G2. While S phase cells were detected by EdU stain, G1 and G2 cells were discerned by the lack of EdU stain and the number of centrin1 signals (centrioles). b IF of cells after 72 h of depletion. In the siCEP44 sample (lower panel) G1 cells contained less centrosomes as judged by the number of γ-tubulin foci (Cenp-F in Supplementary Fig. 1b). c Quantification of b. 80.4 ± 5.0% of G1 cells contained <2 centrosomes. d IF of 60 h siRNA treated RPE1 cells in G1. While the G1 control cells contained 2 defined PCM foci (γ-tubulin) accompanied by equal number of CEP44 foci, in the siCEP44 sample the loss of CEP44 correlated with inefficient PCM (γ-tubulin) recruitment to only one (bottom panel) or both centrosomes (middle panel) (Cenp-F, Supplementary Fig. 1g). e Quantification of CEP44 loss in d. 65.1 ± 7.3% of G1 cells contained <2 CEP44 foci. f Quantification of γ-tubulin defined foci in d. 60.7 ± 4.2% of G1 cells contained <2 defined γ-tubulin signals. g, h G2 CEP44-depleted cells after 60 h of CEP44 depletion showed a mild centriole duplication defect as judged by the counting of CEP97 foci (<4). g Quantification of h. 30.3 ± 4.1% of CEP44-depleted cells contained <4 centrioles. i MT regrowth assay in RPE1 C-Nap1 KO cells. The non-converted daughter centrosome without CEP164 staining regrew lower numbers of MTs (5.1 ± 2.7 MTs/centrosome) than the siControl daughter centrosomes (10.3 ± 3.0 MTs/centrosome) upon cold treatment and MT regrowth. j Quantification of i. k Loss of CEP44 leads to misalignment of mitotic spindles (>65%) generating either bipolar asymmetric spindles (51.3 ± 4.7%) or mono-/multipolar ones (14.3 ± 4.0). l Quantification of k. (a, b, d, h, i, k, scale bars : 10 µm, magnification scale bars : 1 µm; c, e, f, g, j and l data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file).

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CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-gamma Tubulin antibody [TU-30] - Centrosome Marker (AB27074)

Immunocytochemistry-immunofluorescence using Anti-gamma Tubulin antibody [TU-30], ab27074. Publication image from Atorino, E. S. et al., 2020, Nat Commun, 32060285. Legend direct from paper.

The CEP44-POC1B complex is needed to convert centrioles to centrosomes.a Anti-Flag IP using CEP44-Flag from RPE1 cells was analysed for POC1B, POC1A and CEP295 by immuno-blotting (IB). GAPDH was used as input control. b Coomassie Blue stained gel of in vitro binding between purified, recombinant CEP44-Flag and purified, recombinant POC1B-HA. See Supplementary Fig. 5b for IB and 5c for Coomassie Blue stained gels of purified proteins used in the experiment. c Schematic representation of CEP44 protein sequence identity in vertebrata (referred to Supplementary Fig. 5a). d CEP44-Flag constructs that were designed based on c and used in e. e IB of input and eluted samples from RPE1 IPs using CEP44-Flag constructs as outlined in d. The CEP44-Flag IPs were tested for the presence of POC1B. GAPDH was used as loading control for the input. f, g 39.2 ± 2.8% of G1 cells in which POC1B was depleted show <2 γ-tubulin defined foci (Cenp-F in Supplementary Fig. 5g). h–j Loss of either CEP44 or POC1B in response to siRNAs depletion by one of them. h, i CEP44 loss upon CEP44 siRNA has a similar impact on POC1B loss from dCs. i Quantification of h. h, j CEP44 delocalisation was less severe than POC1B loss upon POC1B siRNA. j Quantification of h. Upon siPOC1B depletion, CEP44 delocalised (18.9 ± 4.3% of G1 cells) less sever than POC1B (45.5 ± 2.3%). k Schematic representation of the functional interdependency between the conversion molecules in the CCC mechanism. (f, h, scale bars : 10 µm, magnification scale bars : 1 µm; g, i, j data are presented as mean ± s.d., all statistics were derived from two-tail unpaired t-test analysis of n = 6 biologically independent experiments and source data are provided as a Source Data file).