Journal: bioRxiv

Article Title: Mother centrioles are dispensable for deuterosome formation and function during basal body amplification

doi: 10.1101/373662

Figure Lengend Snippet: (a,b) Experimental design. Depletion of Plk4 is known to abolish centriole duplication in proliferating cells and result in cells with only the old MC (1MC) or no MC (0MC), which could thus be used to examine the contribution of MCs to the deuterosome formation ( a ). We transfected radial glia from mouse brain tissues with lentiviral particles at SS −7 to silence Plk4 expression and examined their progeny cells at SS d0 and d3 ( b ). (c) Confirmation of the Plk4 RNAi efficiency using mEPCs at SS d3. The reduced expression of Deup1 in the Plk4-depleted cells is attributed to reduced multiciliate cell differentiation. (d) Typical cells at SS d0 and d3, immunostained for Zo-1, Cep152, and Cep63, an MC-specific marker. GFP-Centrin1 expressed from the lentiviruses served as both infection and centriole markers. The green fluorescence was enhanced using anti-GFP antibody and Alexa Fluor-488-conjugated secondary antibody. MCs and representative deuterosomes, denoted respectively by arrows and arrowheads, were magnified 2-fold to show details. An illustration is provided for each set of the magnified images. (e) MC contents of the cells examined at SS d0 and d3. The results were from three independent experiments and presented as mean ± SD. At least 186 cells at SS d0 and 80 deuterosome-containing cells at SS d3 were scored in each experiment and condition. Student’s t test: ** P <0.01; *** P <0.001. (f) Confirmation of deuterosome formation in Plk4i-expressing mEPCs at SS d3 by co-staining for Deup1 . (g) Box plots for deuterosome numbers per cell. At least 156 deuterosome-containing cells from three independent experiments were scored. Student’s t test: ns, not significant; *** P <0.001.

Article Snippet: Commercial primary antibodies used were: mouse anti-Sas6 (sc-81431, Santa Cruz; IF 1:50), mouse anti-Centrin (04-1624(20H5), Millipore; IF 1:200), mouse anti-α-Tubulin (T5168, Sigma-Aldrich; WB 1:5000), mouse anti-acetylated tubulin (T7451, Sigma-Aldrich; IF 1:1000), mouse anti-Zo-1 (33-9100, ThermoFisher; IF 1:1000), rabbit anti-Centrin 1 (12794-1-AP, Proteintech; IF 1:200), rat anti-GFP (sc-101536, Santa Cruz; IF 1:50), rabbit anti-GFP (A-11122, ThermoFisher; WB 1:3000), and rabbit anti-Cep63 (06-1292, Millipore; IF 1: 200).

Techniques: Transfection, Expressing, Cell Differentiation, Marker, Infection, Fluorescence, Staining

Journal: bioRxiv

Article Title: Mother centrioles are dispensable for deuterosome formation and function during basal body amplification

doi: 10.1101/373662

Figure Lengend Snippet: (a,b) The remaining MC was positive for centriolar appendage proteins Cep164 and Odf2. Cultured mEPCs treated as described in were immunostained to visualize Zo-1, Cep152, and Cep164 or Odf2. GFP-Centrin1, whose green fluorescence was enhanced using anti-GFP antibody and Alexa Fluor-488-conjugated secondary antibody, served as both infection and centriole markers. MCs and representative deuterosomes, denoted respectively by arrows and arrowheads, were magnified 2-fold to show details. An illustration is provided for each set of the magnified images. (c) The remaining MC was a basal body. mEPCs treated as described in were immunostained to visualize Cep152, GFP, and acetylated tubulin (AC-tub; cilia marker). Note that the bottom region of the cilia is Centrin-positive, which explains why the Centrin-staining of the old MC frequently extends far over the centriolar appendages . Ciliogenesis of the young MC in stage-III control cells also explains why the MC becomes Cep164- and Odf2-positive in .

Article Snippet: Commercial primary antibodies used were: mouse anti-Sas6 (sc-81431, Santa Cruz; IF 1:50), mouse anti-Centrin (04-1624(20H5), Millipore; IF 1:200), mouse anti-α-Tubulin (T5168, Sigma-Aldrich; WB 1:5000), mouse anti-acetylated tubulin (T7451, Sigma-Aldrich; IF 1:1000), mouse anti-Zo-1 (33-9100, ThermoFisher; IF 1:1000), rabbit anti-Centrin 1 (12794-1-AP, Proteintech; IF 1:200), rat anti-GFP (sc-101536, Santa Cruz; IF 1:50), rabbit anti-GFP (A-11122, ThermoFisher; WB 1:3000), and rabbit anti-Cep63 (06-1292, Millipore; IF 1: 200).

Techniques: Cell Culture, Fluorescence, Infection, Marker, Staining

Journal: Oncogenesis

Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

doi: 10.1038/oncsis.2017.47

Figure Lengend Snippet: EGFR degradation is impaired in the absence of flotillin-1.( a , b ) Control siRNA, flotillin-1 (F1-siRNAa and -b) and ( c , d ) control HeLa or flotillin-1-knockout cells (transfected with flotillin-1-EGFP or not) were starved overnight and stimulated with 100 ng/ml EGF for 60 min, fixed with methanol and immunostained for EGFR. ( b ) The amount of undegraded EGFR in flotillin-1-knockdown cells was quantified as fluorescence intensity in arbitrary units using the ImageJ Software. A significant increase of EGFR signal was observed in flotillin-knockdown cells as compared with the control. ( d ) EGFR amount after 60 min was quantified as in c , and the data show a rescue of EGFR degradation upon flotillin-1-EGFP expression (cells marked with *, images for GFP in ). The data in c , d are shown as mean±s.d. Cells from three independent experiments were evaluated (control siRNA cells: n =136, F1-siRNAa: n =135 and F1-siRNAb: n =149; control HeLa: n =358, flotillin-1 KO: n =181, rescue cells: n =252). Statistical analysis was performed with one-way analysis of variance (ANOVA) and Bonferroni post-test in comparison with the control. *** P <0.001. Scale bar in ( a , c , e ): 10 μm. ( e ) Flotillin-1-knockdown cells (F1-siRNAb) were treated as in a and immunostained for EGFR (green) and Rab5, Hrs or LAMP1 (red).

Article Snippet: Mouse monoclonal antibody against Hrs (sc-271455, clone C-7; WB: 1:1000), CD63/LAMP3 (sc-5275; IF: 1:200), ubiquitin (sc-8017, clone P4D1; WB: 1:1000, IF: 1:100) and EGFR (sc-120, clone 528; IF: 1:100, WB: 1:1000) as well as rabbit polyclonal antibodies against Hrs (sc-30221, clone M-79; IF: 1:40, WB: 1:1000; IP: 1:100), Rab5 (sc-46692; IF: 1:200) and c-myc (sc-789; IP: 1:300) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Knock-Out, Transfection, Knockdown, Fluorescence, Software, Expressing, Comparison

Journal: Oncogenesis

Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

doi: 10.1038/oncsis.2017.47

Figure Lengend Snippet: Endosomal morphology is altered in flotillin-1-knockdown/knockout cells, but endosomal domains persist.( a ) Control and flotillin-1-knockout cells were chased for 2 h with Fe-EGF, fixed and analysed by transmission electron microscopy. Note the enlarged endosomes in flotillin-1-knockdown cells. Scale bar: 500 nm. ( b ) Control siRNA and flotillin-1-knockdown cells were stimulated with EGF for 30 min and labelled with an anti-mouse LAMP3 antibody detected with a secondary Alexa Fluor 647-coupled antibody. The specimens were analysed with Leica SR GSD 3D microscope. Scale bar: 2 μm. ( c ) Rab5-Q79L-GFP-expressing HeLa cells were immunostained for endogenous flotillin-2 and Hrs, which show a limited colocalization in the enlarged endosomes. Scale bar: 10 μm. ( d ) Rab5-Q79L-GFP-expressing HeLa and flotillin-1-knockout cells were immunostained for endogenous CHC and Hrs, which are localised in endosomal domains in both cell types and show some colocalization. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibody against Hrs (sc-271455, clone C-7; WB: 1:1000), CD63/LAMP3 (sc-5275; IF: 1:200), ubiquitin (sc-8017, clone P4D1; WB: 1:1000, IF: 1:100) and EGFR (sc-120, clone 528; IF: 1:100, WB: 1:1000) as well as rabbit polyclonal antibodies against Hrs (sc-30221, clone M-79; IF: 1:40, WB: 1:1000; IP: 1:100), Rab5 (sc-46692; IF: 1:200) and c-myc (sc-789; IP: 1:300) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Knock-Out, Control, Transmission Assay, Electron Microscopy, Microscopy, Expressing