Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a ) LC3-II levels in HeLa cells transfected with siRNA against scrambled control or CCT subunits treated with vehicle (DMSO) or Bafilomycin A1 (400 nM) for 5 h. GAPDH is loading control. ( b ) LC3-II/GAPDH densitometry from a . Graphs show means±s.d. of triplicate samples in the absence or presence of Bafilomycin A1. Similar results were observed in at least three independent experiments. ( n =3; ** P <0.01, * P <0.05; two-tailed one sample t -test). For combined data of independent biological replicates, see . ( c ) LC3-II levels in mouse primary cortical neurons transduced with two different shRNA lentiviral particles targeting either control (Ctrl sh#1 is empty vector and Ctrl sh#2 is control scramble) or the mouse Cct5 (Cct5 sh#1 and Cct5 sh#2). Cells were treated with vehicle (DMSO) or Bafilomycin A1 at 200 nM for 12 h. GAPDH is loading control. ( d ) LC3-II/GAPDH densitometry from c . Graphs show mean±s.d. of triplicate samples in the absence or presence of Bafilomycin A1. Similar results were observed in another two independent experiments. In Bafilomycin A1 conditions, the Cct5 shRNAs (#1 or #2) treated cells showed significant reduced LC3-II levels when compared with the scramble control (Ctrl sh#2) treated neurons ( n =3; * P <0.05, NS, not significant; two-tailed t -test), but not-significant if compared with the empty-vector control (Ctrl sh#1) condition ( n =3; NS, not significant; two-tailed one sample t -test). ( e ) GFP dot numbers (autophagosomes/non-acidified autolysosomes, mRFP-GFP double labelled vesicles) and mRFP-only dots (acidified autolysosomes) in HeLa cells. HeLa cells stably expressing mRFP-GFP-LC3 were treated as in a then seeded in a 96-well plate and subjected to automated Cellomics visualization and analysis. Graph shows results from one representative experiment. We analysed 100–200 cells per well (from a 96-well plate) with 10–12 wells per condition. Similar data were seen in other two independent experiments. Bars—means±s.e.m. (*** P <0.001; two-tailed t -test). ( f ) Representative confocal z-stack images (left panel) and total number of GFP dots (autophagosomes) and mRFP-only dots (autolysosomes)—left panel, in mouse primary cortical neurons from mRFP-GFP-LC3 transgenic mice. Cortical neurons were plated in live cell dishes, treated as in d and the numbers of dots were manually counted after confocal microscopy. Graph shows data from three independent biological replicates; 100–150 neurons counted per each condition. Bars—means±s.e.m. (*** P <0.001; two-tailed t -test). Scale bar—10 μm.

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Transfection, Control, Two Tailed Test, Transduction, shRNA, Plasmid Preparation, Stable Transfection, Expressing, Transgenic Assay, Confocal Microscopy

Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a ) LC3 (anti-mouse Alexa 488) and LAMP1 (anti-rabbit Alexa 568) colocalization in HeLa cells depleted for CCT5. Right panel: quantification of colocalization (Pearson's coefficient). More than 30 cells were counted per condition. Bars—means±s.e.m. (*** P <0.001, * P <0.05, NS, not significant; two-tailed t -test). Similar results were achieved in at least two independent experiments. Scale bar, 10 μm. ( b ) Immunoblot showing Cathepsin L (CTSL) maturation in CCT5 knockdown HeLa cells. Graph showing the quantification of mature/precursor CTSL ratio is on right as mean±s.d. ( n =3; ** P <0.01, * P <0.05; two-tailed one sample t -test). ( c ) Cathepsin L activity in HeLa cells depleted for CCT5. Fluorescence intensity was expressed as percentage of control. Bars—mean±s.d. ( n =3; ** P <0.01; two-tailed t -test). ( d ) Cathepsin D (CTSD, anti-mouse Alexa 488)—LAMP1 (anti-rabbit Alexa 568) and cathepsin D (anti-mouse Alexa 488) —calnexin (anti-rabbit Alexa 568) double-immunostaining in CCT5-knockdown cells. More than 40 cells were quantified per experiment (each experiment was performed at least twice with similar results). Bars—mean of Pearson's coefficient±s.e.m. (*** P <0.001; two-tailed t -test). Scale bar—10 μm. See also . ( e ) Lysosomal pH in CCT-depleted cells. The Lysosensor Yellow Blue intensity was measured in both yellow and blue channels. Intensities ratio is a qualitative measure of lysosomal pH. Bafilomycin A1 (Baf A1) was used as a positive control (400 nM, 3 h) for increased lysosomal pH >50 cells were analysed per experiment, for each condition. Experiment was performed at least 3 times with similar results. Bars—mean±s.e.m. (*** P <0.001; two-tailed t -test). ( f ) Lysosomal pH in Cct5-depleted mouse primary cortical neurons. Lysosensor Yellow Blue intensity was measured as in e . Bafilomycin A1 was used as a positive control (400 nM, 6 h) for increased lysosomal pH >40 neurons were analysed per experiment and per condition. Experiment was performed at least three times with similar results. Bars—means±s.e.m. (*** P <0.001; two-tailed t -test). See also . ( g ) Voa1 (anti-rabbit Alexa555) and Lamp1 (anti-rat Alexa647) double-immunostaining in mouse primary cortical neurons. Neurons were plated and treated as in f . More than 30 neurons were counted per condition. PC is Pearson's coefficient; M1 is Mander's coefficient 1, which indicates the Voa1 localization in the Lamp1 compartment. Bars—means±s.e.m. (*** P <0.001; two-tailed t -test). Scale bar—10 μm. See also .

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Two Tailed Test, Western Blot, Knockdown, Activity Assay, Fluorescence, Control, Double Immunostaining, Positive Control

Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a ) LC3-II levels in HeLa cells treated with latrunculin A (1 μM) or cytochalasin D (1 μM) for 3 h in the presence or absence of Bafilomycin A1 (400 nM, 3 h). ( b ) LC3-II/GAPDH densitometry in HeLa cells treated with Latrunculin A or Cytochalasin D as in a . Bars—means±s.d. ( n =3; ** P <0.01; * P <0.05; two-tailed one sample t -test). ( c ) GFP dot numbers (autophagosomes) and mRFP-only dots (autolysosomes) in HeLa cells stably expressing mRFP-GFP-LC3 and independently treated with 1 μM latrunculin A or cytochalasin D for 3 h. Graphs show the results for one representative experiment: eight wells of 100–200 cells each were analysed per sample. Experiment was repeated with similar results. Bars—means±s.e.m. ( n =8; ** P <0.01, * P <0.05; two-tailed t -test) ( d ) Cathepsin L (CTSL) activity in HeLa cells treated with the indicated drugs (1 μM, 3 h). Bafilomycin was positive control (400 nM, 3 h). Bars—means±s.d. ( n =3; *** P <0.001, ** P <0.01; two-tailed t -test). ( e ) Cathepsin D (CTSD, anti-mouse Alexa 488) and LAMP1 (anti-rabbit Alexa 568) double-immunostaining in cells treated with latrunculin A (1 μM, 3 h). Bottom graph—colocalization quantification. More than 30 cells were counted per experiment (each experiment was performed at least twice). Bars—mean±s.e.m. (*** P <0.001; two-tailed t -test). Scale bar, 10 μm. ( f ) Cathepsin D (anti-mouse Alexa 488) and calnexin (anti-rabbit Alexa 568) double-immunostaining in latrunculin A-treated cells (1 μM, 3 h). Bottom panel—colocalization quantification. More than 30 cells were counted per experiment (each experiment was performed at least twice). Bars—means±s.e.m. (*** P <0.001; two-tailed t -test). Scale bar—10 μm. ( g ) Lysosomal pH in cells treated with actin disrupting drugs (latrunculin A, cytochalasin D; 3 h, 1 μM) in full medium. Bafilomycin A1—positive control for increased lysosomal pH. More than 50 cells were counted per experiment, performed twice. Bars—means±s.e.m., where DMSO control was set up to 1 (arbitrary units) (*** P <0.001, * P <0.05; two-tailed t -test). ( h – j ) mRFP-LC3 and Lgp120-GFP live-cell imaging in HeLa cells exposed to Latrunculin A (1 μM). Autophagosome–lysosome fusion is not affected compared with the control. Scale bar, 10 μm. ( i ) mRFP-LC3 and Lgp120-GFP colocalization expressed as Pearson's coefficient. More than 25 cells were counted per experiment. Bars—means±s.e.m. (NS, not significant; two-tailed t -test). ( j ) Autophagosome–lysosome fusion events in HeLa cells expressing mRFP-LC3 and Lgp120-GF. For both control and Latrunculin A treatment, the movies show five frames compressed per second (from a total of 70 frames, taken continuously). Arrows—fusion events. Scale bar, 10 μm.

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Two Tailed Test, Stable Transfection, Expressing, Activity Assay, Positive Control, Double Immunostaining, Control, Live Cell Imaging

Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a ) LC3-II blots of HeLa cells treated with CCT5 siRNA and exposed to latrunculin A (1 μM, 3 h). ( b ) LC3-II/GAPDH densitometry in HeLa cells treated as in a . Bars—means±s.d. ( n =3; * P <0.05, NS, not significant; two-tailed one sample t -test). ( c ) In vitro cathepsin L (CTSL) activity in HeLa cells exposed to CCT5 siRNA. Fluorescence intensity expressed as percentage of control. The cells were additionally exposed to Latrunculin A (3 h, 1 μM). Bars—means±s.d. ( n =3; *** P <0.001, ** P <0.01, NS, not significant; two-tailed t -test). ( d ) In vitro cathepsin L activity in HeLa cells exposed to CCT2 or CCT7 siRNAs. Fluorescence intensity was expressed as percentage of control. The cells were additionally exposed to Latrunculin A (3 h, 1 μM). Bars—means±s.d. ( n =3; *** P <0.001, ** P <0.01, NS, not significant; two-tailed t -test). ( e ) LC3-II levels in HeLa cells exposed to two rounds of PFD6 (prefoldin) smart-pool siRNA treatments in the presence/absence of Bafilomycin A1 (400 nM, 3 h). ( f ) LC3-II/GAPDH densitometry for HeLa cells exposed to two rounds of PFD6 (prefoldin) smart-pool siRNA and treated as in e . Bars—means±s.d. ( n =3; * P <0.05, NS, not significant; two-tailed one sample t -test). ( g ) LC3-II blots of HeLa cells treated with individual PFD6 siRNA oligos in presence/absence of Bafilomycin A1 (400 nM, 5 h). LC3-II/GAPDH ratio is displayed relative to control. ( h ) Cathepsin L activity in HeLa cells treated as in e . Bafilomycin A1 was positive control (400 nM, 3 h). Bars—means±s.d. ( n =3; *** P <0.001, ** P <0.01, NS, not significant; two-tailed t -test). ( i ) Number and size of GFP dots (autophagosomes) and mRFP-only dots (autolysosomes). HeLa cells stably expressing mRFP-GFP-LC3 were treated with two rounds of siRNA (smart-pool PFD6 and two individual oligos), then seeded in a 96-well plate and subjected to Cellomics visualization analysis. Graphs show the results for one representative experiment: 10 wells of 100–200 cells each were analysed per sample. Similar data was seen in other two independent experiments. Bars—means±s.e.m. ( n =10; *** P <0.001, ** P <0.01, * P <0.05; two-tailed t -test).

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Two Tailed Test, In Vitro, Activity Assay, Fluorescence, Control, Positive Control, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a ) EGFP-HTT(Q74) aggregation and cell toxicity in CCT-depleted HeLa cells. Upper: HeLa cells ( ATG16L1+ ) were seeded on coverslips in triplicates, transfected with siRNAs targeting control or individual CCT subunits, followed by 1 μg of EGFP-HTT(Q74) overexpression. Bottom: percentage of nuclei with morphological changes (fragmented or condensed nuclei). Bars—means of percentages of cells with aggregates±s.d. ( n =3; ** P <0.01; * P <0.05; two-tailed t -test). Similar results were observed in four independent experiments. See for representative images. ( b ) EGFP-HTT(Q74) aggregation and cell toxicity in ATG16L1− CRISPR/Cas9 HeLa cells. The experiment was performed in parallel with a . Bars—means of percentages of cells with aggregates±s.d. ( n =3; NS, not significant; two-tailed t -test). Similar results were observed in four independent experiments. See for representative images. ( c ) EGFP-HTT(Q74) aggregation and cell toxicity in WT and Atg16L1 −/− MEFs. Cells were seeded on coverslips in triplicates, transduced with lentiviral particles targeting control or the mouse Cct5 subunit (two independent shRNAs) and transfected with EGFP-HTT(Q74). Bars—means of percentages of cells with aggregates±s.d. ( n =3; *** P <0.001, ** P <0.01, * P <0.05, NS, not significant; two-tailed t -test). Similar data was achieved in 3 independent experiments. See for representative images. ( d ) N17-103Q-GFP aggregation in CCT-depleted ATG16L1+ and ATG16L1− HeLa cells. Representative images of N17-103Q-GFP aggregates are displayed on right. Bars—mean of the percentages of cells with aggregates±s.e.m. ( n =4 independent experiments, each performed in triplicates; ** P <0.01, NS, not significant; one sample t -test). ( e ) N17-97QP-GFP aggregation in CCT-depleted ATG16L1+ and ATG16L1− HeLa cells. Representative images of N17-103Q-GFP aggregates are on right. Bars—mean of the percentages of cells with aggregates±s.e.m. ( n =4 independent experiments, each performed in triplicates; ** P <0.01, NS, not significant; one sample t -test). ( f ) CCT co-immunoprecipitation with EGFP-HTT(Q74). HeLa cells were transfected with either GFP-vector or EGFP-HTT(Q74) for 16 h and GFP-tagged proteins were pulled-down with GFP-Trap. ( g ) CCT co-immunoprecipitation with N17-103Q-GFP and N17-97QP-GFP. HeLa cells were transfected with GFP-vector, N17-103Q-GFP or N17-97QP-GFP for 16 h and GFP-tagged proteins were pulled-down with GFP-Trap. Note: N17-103Q-GFP does not have the proline-rich domain, while N17-97QP-GFP has both N terminus and proline-rich domains of htt exon 1.

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Transfection, Control, Over Expression, Two Tailed Test, CRISPR, Transduction, Immunoprecipitation, Plasmid Preparation

Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a , b ) Oligomerization of mutant htt in CCT5-depleted Hela cells, both ATG16L1+ and ATG16L1−. CCT5 depletion enhanced the amount of coimunoprecipitation of GFP-tagged mutant HTT(1–548) with Flag-tagged mutant HTT(1–588) —see left a and vice-versa —see right b , in autophagy competent HeLa cells, but failed to do so in the autophagy incompetent cells: ATG16L1− HeLa. ( c ) Quantification of mutant htt co-immunoprecipitation relative to total lysates/input from three independent experiments. The control condition was set to 100 and the bars represent the mean±s.d. ( n =3; * P <0.05, NS, not significant; two-tailed sample t -test). See Methods. ( d ) CCT co-immunoprecipitation with the Htt(1–548)Q15 and Htt(1–548)Q138 constructs tagged with GFP at the C terminus. HeLa cells were transfected with GFP-vector, Htt(1–548)Q15 –GFP or Htt(1–548)Q138-GFP for 48 h and the GFP-tagged proteins were pulled-down using the GFP-Trap technology. ( e ) 3B5H10 staining in CCT5-depleted HeLa cells, both ATG16L1+ and ATG16L1−. Scale bar throughout the panel is 10 μm. See Methods. ( f ) Quantification of 3B5H10 to Flag ratio signal intensities from three independent experiments. The bars represent the mean±s.d. ( n =3; * P <0.05, NS, not significant; two-tailed sample t -test). ( g ) 3B5H10 antibody preferentially label cells overexpressing the pathogenic polyQ expansions in Htt(1–588). HeLa cells were transiently transfected with either Flag-tagged HTT(1–588)Q138 or Flag-tagged HTT(1–588)Q15 and immunostained with 3B5H10 antibody. Scale bar throughout the panel is 10 μm.

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Mutagenesis, Immunoprecipitation, Control, Two Tailed Test, Construct, Transfection, Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: CCT complex restricts neuropathogenic protein aggregation via autophagy

doi: 10.1038/ncomms13821

Figure Lengend Snippet: ( a ) GFP-ATXN3(Q84) aggregation in CCT-depleted ATG16L1+ and ATG16L1- HeLa cells. The bars represent the mean of the percentages of cells with aggregates±s.e.m. ( n =4 independent experiments, each performed in triplicates; *** P <0.001, NS, not significant; one sample t -test). ( b ) Representative images of GFP-ATXN3(Q84) aggregates for the experiments performed in a . Scale bar throughout the panel is 10 μm. ( c ) EGFP-A19 aggregation in WT and Atg16L1 −/− MEFs. Cells seeded on coverslips in triplicates, were transduced with two different shRNA lentiviral particles targeting either control or the mouse Cct5 subunit and transfected with EGFP-A19. The bars represent the mean of percentages of cells with aggregates±s.d. ( n =3; * P <0.05, NS, not significant; two-tailed t -test). Similar data was achieved in other two independent experiments. ( d ) CCT co-immunoprecipitation with the GFP-ATXN3(Q84), GFP-p62 (on the left ) and EGFP-A19, N17-97QP-GFP (on the right). HeLa cells were transfected with GFP-vector or the above mentioned proteins tagged to GFP for 16 h; the GFP-tagged proteins were pulled-down using the GFP-Trap technology. ( e ) Western blotting for p62. ATG16L1+ and ATG16L1− CRISPR/Cas9 HeLa cells were transfected with siRNA targeting individual CCT subunits. GAPDH was used as loading control. ( f ) Quantification of p62 accumulation. ATG16L1+ and ATG16L1− CRISPR/Cas9 HeLa cells were transfected with siRNA targeting individual CCT subunits as in ( e ). GAPDH was used as loading control. The bars represent the mean±s.d. ( n =3; ** P <0.01, * P <0.05, NS, not significant; two-tailed one sample t -test). Similar results were observed in three independent experiments.

Article Snippet: HeLa cells (source ATCC) were authenticated by STR profiling.

Techniques: Transduction, shRNA, Control, Transfection, Two Tailed Test, Immunoprecipitation, Plasmid Preparation, Western Blot, CRISPR