Journal: Nature Communications

Article Title: Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging

doi: 10.1038/s41467-023-42822-1

Figure Lengend Snippet: a Schematic of crossing scheme used to obtain mice with APLP2 knockout and neuronal conditional deletion of APP , termed N-dCKO. Created with BioRender.com. b Representative immunofluorescent images show increased ubiquitin (red) immunostaining in N-dCKO neurons (MAP2, green), additionally stained for DAPI (blue), compared to controls. c Quantitative analysis shows significantly increased ubiquitin levels in N-dCKO neurons compared to controls. Control n = 45, App KO n = 45. p -value = 2.61E-08. d Representative confocal images show increased staining for LC3B (red), a marker of autophagy, in N-dCKO neurons (NeuN, green) compared to controls. e Quantitative analysis shows a significant increase in LC3B intensity in N-dCKO neurons compared to controls. Control n = 90, App KO n = 100. p -value = 4.98E-11. f Representative immunofluorescent images using an antibody specific for phosphorylated SMAD3, a TGFβ transcription factor, show decreased phospho-SMAD3 (red) in N-dCKO neurons (NeuN, green) compared to controls. g Quantification shows significantly decreased phospho-SMAD3 in N-dCKO neurons compared to controls. Control is APLP2ˉ / ˉ . Control n = 136, App KO n = 141. p -value = 1.06E-08. **** p < 0.0001, two-tailed Student’s t-test. Data are represented as mean ± SEM. n = 5 mice per genotype. Scale bars are 5 μm. Mice are 18 months old. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies to the following proteins were used at the indicated concentrations: ubiquitin (1:1000, P4G7, BioLegend; 1:200 Cell Signaling; 1:200, Z0458, Dako), GABARAP (1:1000, endogenous Drosophila Atg8a, E1J4E, Cell Signaling; 1:200, Abcam), LC3B (1:200, Novus Biologicals), ref(2)P (1:600, Sarkar et al. ,), Rab5 (1:100, ab31261, Abcam), Rab11 (1:100, 610656, BD Biosciences), GFP (1:200, N86/8, NeuroMab), cleaved PARP (1:5000, E51, Abcam), elav (1:5, 9F8A9, Developmental Studies Hybridoma Bank), NeuN (1:400, EMD Millipore), MAP2 (1:100, EMD Millipore), pSMAD3 (1:200, Abcam).

Techniques: Knock-Out, Immunostaining, Staining, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging

doi: 10.1038/s41467-023-42822-1

Figure Lengend Snippet: a Schematic of the approach to generate APP knockout and isogenic control neurons. Created with BioRender.com. b Representative immunofluorescence images show increased ubiquitin (red) immunostaining in APP knockout neurons (MAP2, magenta), additionally stained for DAPI (blue), compared to controls. c Quantitative analysis shows significantly increased ubiquitin levels in APP knockout neurons compared to controls. Control n = 133, APP KO n = 151. p -value = 8.85E-09. d Immunostaining shows increased LC3B (arrow, red) immunostaining in APP knockout neurons (NeuN, magenta) compared to controls. e Quantitative analysis shows significantly increased LC3B levels in APP knockout neurons compared to controls. Control n = 17, APP KO n = 19. p -value = 3.86E-08. f Immunostaining shows decreased phospho-SMAD3 (red) immunostaining in APP knockout neurons (NeuN, magenta) compared to controls. g Quantitative analysis shows significantly decreased phospho-SMAD3 levels in APP knockout neurons compared to controls. Control n = 150, APP KO n = 156. p -value = 9.76E-11. *** p < 0.0001, two-tailed Student’s t-test. Data are represented as mean ± SEM. Cells from 3 independent differentiations of APP knockout and isogenic control cells were analyzed. Scale bars are 10 μm. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies to the following proteins were used at the indicated concentrations: ubiquitin (1:1000, P4G7, BioLegend; 1:200 Cell Signaling; 1:200, Z0458, Dako), GABARAP (1:1000, endogenous Drosophila Atg8a, E1J4E, Cell Signaling; 1:200, Abcam), LC3B (1:200, Novus Biologicals), ref(2)P (1:600, Sarkar et al. ,), Rab5 (1:100, ab31261, Abcam), Rab11 (1:100, 610656, BD Biosciences), GFP (1:200, N86/8, NeuroMab), cleaved PARP (1:5000, E51, Abcam), elav (1:5, 9F8A9, Developmental Studies Hybridoma Bank), NeuN (1:400, EMD Millipore), MAP2 (1:100, EMD Millipore), pSMAD3 (1:200, Abcam).

Techniques: Knock-Out, Immunofluorescence, Immunostaining, Staining, Two Tailed Test

Journal: Nature

Article Title: MYCT1 controls environmental sensing in human haematopoietic stem cells.

doi: 10.1038/s41586-024-07478-x

Figure Lengend Snippet: Fig. 4 | MYCT1 is located in endosomes and interacts with vesicle trafficking and receptor signalling machinery. a, The transmembrane topology of MYCT1 was predicted from its amino acid sequence using Phobius48 and is depicted as a schematic and histogram of probability. b,c, High-resolution Airyscan immunofluorescence images (b) and quantification (c) of the localization of overexpressed MYCT1–V5 in human CB HSPCs after 5 days of culture. MYCT1–V5 was visualized by staining for V5, and colocalization with endosomal markers (clathrin, RAB5, RAB7 and RAB11), Golgi marker (GM130) and mitochondrial marker HSP60 was evaluated. DAPI indicates nuclei. Colocalization channel (Coloc) shows areas positive for V5 and each marker. Scale bars, 3 µm (RAB5) or 2 µm (other columns). Analysis was performed using Imaris (v.9.7.2) software

Article Snippet: HSP60 D6F1 Cell Signaling Technology 12165T, reported recognize endogenous human HSP60 (manufacturer’s website) Lamin B1 EPR8985(B) Abcam ab133741, reported recognize human LaminB1 (manufacturer’s website) Na/K ATPase EP1845Y Abcam ab76020, reported recognize human Na/K ATPase (manufacturer’s website) phospho-AKT D9E Cell Signaling Technology 4060S, reported recognize endogenous human pAKT (Ser 473) (manufacturer’s website) phospho-ERK D13.14.4E Cell Signaling Technology 4370S, reported recognize endogenous human pERK1/2 (Thr202/Tyr204) (manufacturer’s website) Rab11 D4F5 Cell Signaling Technology 5589T, reported recognize endogenous human Rab11 (manufacturer’s website) Rab5 C8B1 Cell Signaling Technology 3547T, reported recognize endogenous human Rab5 (manufacturer’s website) Rab7 D95F2 Cell Signaling Technology 9367T, reported recognize endogenous human Rab7 (manufacturer’s website) V5 SV5-Pk1 ThermoFisher Scientific R960-25, reported recognize V5 tag (manufacturer’s website).

Techniques: Sequencing, Immunofluorescence, Staining, Marker, Software