Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

doi: 10.1155/2020/8908901

Figure Lengend Snippet: The CM and ALA-CM pretreatment reversed Amyloid β - (A β 1-42 -) induced synaptic toxicity in differentiated SH-SY5Y cells. On the day 6th, the SH-SY5Y cells (differentiated) were pretreated for 1 h with CM or ALA-CM before the addition of 5 μ M A β 1-42 for the next 24 h. The SH-SY5Y cells were also exposed to the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence in CM and ALA-CM was responsible for the neuroprotective effect. Positive controls were the SH-SY5Y cells treated with insulin and carbonyl-cyano-m-chlorophenylhydrazone-CCCP (10 μ M). RT-qPCR results indicated that A β 1-42 significantly decreased mRNA levels of Synaptophysin (a) and PSD95 (b), well-known synaptic markers. The CM and ALA-CM pretreatment reversed the effect of A β 1-42 when compared with DM + A β 1−42 group. The IDE treatment of CM and ALA-CM reduced this effect. The immunocytofluorescence staining showed that the A β 1-42 treated cells had a decreased Synaptophysin (c, e) and TUJ 1 ( β 3-Tubulin) (d, f) fluorescence intensity and increased neurites fragmentation. Moreover, results showed that the CM and ALA-CM pretreatment reversed the A β 1-42 –induced synaptic toxicity in differentiated SH-SY5Y cells. A similar effect of TUJ 1 fluorescence intensity was observed after the treatment of differentiated SH-SY5Y cells with insulin (d, f). The IDE treatment of CM and ALA-CM reduced this effect. The cells were subjected to immunocytofluorescence staining with antibodies against Synaptophysin and TUJ 1. TUJ 1 was used as a marker to stain differentiated SH-SY5Y cells (show as green signals). Synaptophysin was used to stain synaptic in differentiated SH-SY5Y cells (show as red signals). Hoechst 33342 was used to stain nuclei (show as blue signals) (see Materials and Methods section). Bar graphs (e, f) showed the relative fluorescence intensity of Synaptophysin and TUJ 1. Scale bar is 20 μ m. One-way ANOVA followed by Tukey's multiple comparisons test at the 0.05 level was used to determine differences between the treated cells and untreated control cells. Results are presented as means ± SEM ( n = 3 − 8). RT-qPCR fold increase and the fluorescence intensity were calculated according to the formula described in the Materials and Methods section. Statistical differences between the treated group and untreated control cells are indicated by asterisks ( ∗ for P < 0.05; ∗∗ for P < 0.01; ∗∗∗ for P < 0.001; # ∗∗∗ versus the control group; ## ∗∗∗ versus DM + A β 1−42 group).

Article Snippet: Next, the cells were incubated with mouse monoclonal anti- β 3 Tubulin antibody (TUJ 1; Santa Cruz Biotechnology, Inc.; 1: 50 in 1% BSA-PBS, for 1 h, RT), rabbit anti-Synaptophysin (GeneTex, Inc.; 1 : 500 in 1% BSA-PBS, for 1 h, RT), mouse anti-PARKIN (Abcam Inc.; 1 : 200 in 1% BSA-PBS, for 1 h, RT), rabbit anti-TOMM20 antibody (Abcam Inc.; 1 : 500 in 1% BSA-PBS, for 1 h, RT), and Alexa FluorTM488-conjugated donkey anti-mouse IgG or Alexa FluorTM568-conjugated goat anti-rabbit IgG (Invitrogen, Life Technologies; 1 : 200 in 1% BSA-PBS, for 1 h, RT).

Techniques: Quantitative RT-PCR, Staining, Fluorescence, Marker, Control

Journal: Nature Communications

Article Title: CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion

doi: 10.1038/s41467-020-14767-2

Figure Lengend Snippet: 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. ). 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. ). 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).

Article Snippet: Other primary antibodies directed against the indicated proteins were: γ-tubulin (mouse, 1:1000, abcam Ab27074), PCNT (rabbit, 1:2000, abcam Ab4448), CEP97 (rabbit, 1:300, Bethyl A301-945A), Centrin1 (mouse 1:1000, Millipore MABC544), α-tubulin (mouse, 1:500, SigmaAldrich DM1A), SASS-6 (mouse, 1:50, SCBT sc-81431), Flag tag (mouse, IF 1:1600 - WB 1:1000, Cell signaling 9A3), CEP295 (rabbit, 1:500, Abcam Ab122490), GAPDH (rabbit, WB 1:1000, 14C10), HA tag (rat, 1:1000, Böhringer Mannheim No.1867423), GT335 (mouse, 1:500, AdipoGen AG-2013-0020), γ-tubulin (guinea pig, 1:50), CEP135 (rabbit, 1:100), C-Nap1 (goat, 1:1000), CEP164 (rat, 1:2000), CEP152 (rabbit, 1:500, Dr. Ingrid Hoffmann, DKFZ), CEP192 (rabbit, 1:2000, Nigg) and Cenp-F (sheep, 1:1000, Dr. Stephen Taylor, Manchester).

Techniques: Staining, IF-cells, Regrowth Assay, Derivative Assay

Journal: Nature Communications

Article Title: CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion

doi: 10.1038/s41467-020-14767-2

Figure Lengend Snippet: 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. 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. ). 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. ). 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).

Article Snippet: Other primary antibodies directed against the indicated proteins were: γ-tubulin (mouse, 1:1000, abcam Ab27074), PCNT (rabbit, 1:2000, abcam Ab4448), CEP97 (rabbit, 1:300, Bethyl A301-945A), Centrin1 (mouse 1:1000, Millipore MABC544), α-tubulin (mouse, 1:500, SigmaAldrich DM1A), SASS-6 (mouse, 1:50, SCBT sc-81431), Flag tag (mouse, IF 1:1600 - WB 1:1000, Cell signaling 9A3), CEP295 (rabbit, 1:500, Abcam Ab122490), GAPDH (rabbit, WB 1:1000, 14C10), HA tag (rat, 1:1000, Böhringer Mannheim No.1867423), GT335 (mouse, 1:500, AdipoGen AG-2013-0020), γ-tubulin (guinea pig, 1:50), CEP135 (rabbit, 1:100), C-Nap1 (goat, 1:1000), CEP164 (rat, 1:2000), CEP152 (rabbit, 1:500, Dr. Ingrid Hoffmann, DKFZ), CEP192 (rabbit, 1:2000, Nigg) and Cenp-F (sheep, 1:1000, Dr. Stephen Taylor, Manchester).

Techniques: Staining, In Vitro, Binding Assay, Purification, Recombinant, Sequencing, Construct, Functional Assay, Derivative Assay

Journal: Nature Communications

Article Title: CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion

doi: 10.1038/s41467-020-14767-2

Figure Lengend Snippet: 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 with α-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 with α-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 for α-tubulin and Flag. Supplementary Fig. 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. . 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.

Article Snippet: Other primary antibodies directed against the indicated proteins were: γ-tubulin (mouse, 1:1000, abcam Ab27074), PCNT (rabbit, 1:2000, abcam Ab4448), CEP97 (rabbit, 1:300, Bethyl A301-945A), Centrin1 (mouse 1:1000, Millipore MABC544), α-tubulin (mouse, 1:500, SigmaAldrich DM1A), SASS-6 (mouse, 1:50, SCBT sc-81431), Flag tag (mouse, IF 1:1600 - WB 1:1000, Cell signaling 9A3), CEP295 (rabbit, 1:500, Abcam Ab122490), GAPDH (rabbit, WB 1:1000, 14C10), HA tag (rat, 1:1000, Böhringer Mannheim No.1867423), GT335 (mouse, 1:500, AdipoGen AG-2013-0020), γ-tubulin (guinea pig, 1:50), CEP135 (rabbit, 1:100), C-Nap1 (goat, 1:1000), CEP164 (rat, 1:2000), CEP152 (rabbit, 1:500, Dr. Ingrid Hoffmann, DKFZ), CEP192 (rabbit, 1:2000, Nigg) and Cenp-F (sheep, 1:1000, Dr. Stephen Taylor, Manchester).

Techniques: Imaging, Staining, Purification, Binding Assay, Recombinant, Mutagenesis, Incubation, Centrifugation, Construct, Generated, Derivative Assay

Journal: Nature Communications

Article Title: CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion

doi: 10.1038/s41467-020-14767-2

Figure Lengend Snippet: 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. ) 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. ) 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.

Article Snippet: Other primary antibodies directed against the indicated proteins were: γ-tubulin (mouse, 1:1000, abcam Ab27074), PCNT (rabbit, 1:2000, abcam Ab4448), CEP97 (rabbit, 1:300, Bethyl A301-945A), Centrin1 (mouse 1:1000, Millipore MABC544), α-tubulin (mouse, 1:500, SigmaAldrich DM1A), SASS-6 (mouse, 1:50, SCBT sc-81431), Flag tag (mouse, IF 1:1600 - WB 1:1000, Cell signaling 9A3), CEP295 (rabbit, 1:500, Abcam Ab122490), GAPDH (rabbit, WB 1:1000, 14C10), HA tag (rat, 1:1000, Böhringer Mannheim No.1867423), GT335 (mouse, 1:500, AdipoGen AG-2013-0020), γ-tubulin (guinea pig, 1:50), CEP135 (rabbit, 1:100), C-Nap1 (goat, 1:1000), CEP164 (rat, 1:2000), CEP152 (rabbit, 1:500, Dr. Ingrid Hoffmann, DKFZ), CEP192 (rabbit, 1:2000, Nigg) and Cenp-F (sheep, 1:1000, Dr. Stephen Taylor, Manchester).

Techniques: Generated, Staining, Derivative Assay