Fig 1: Her2 stability and protein levels are affected by ATG9A levels(A) Protein abundance of ATG9A, Her2, AKT and P27/KIP upon transfection with ATG9A or empty vector in the presence or absence of trastuzumab (20 µg/ml) in BT474-TR and BT474 (B) mRNA expression levels for ATG9A and Her2 upon transfection with ATG9A or empty vector in the presence or absence of trastuzumab (20 µg/ml) in BT474-TR and BT474. (C) BT474-TR and BT474 cells were treated with Cyclohexamide (100 µM) for up to 48 hours after overexpression of ATG9A or empty vector in the presence or absence of trastuzumab (20 µg/ml). Half-life of Her2 protein (t1/2) was calculated and compared between ATG9A and empty vector groups. (D) Protein abundance for ATG9A, Her2, AKT and (E) mRNA expression levels for ATG9A and Her2 in JIMT-1 and MDA-453 after transfection with ATG9A or empty vector in the presence or absence of trastuzumab (20 µg/ml). (F) JIMT1 and MDA-453 cells were treated with Cyclohexamide (100 µM) for up to 48 hours after overexpression of ATG9A or empty vector in the presence or absence of trastuzumab (20 µg/ml). Half-life of Her2 protein (t1/2) was calculated and compared between ATG9A and empty vector groups.
Fig 2: Prostatic epithelial cell and stromal fibroblast recombination under the renal capsule in nude mice.a, b Recombinant grafts in each group. The shATG9A group (WPMY-shATG9A+BPH-1) showed reduced volumes of recombinant grafts compared with the control group (WPMY-shNC+BPH-1). c, d Immunofluorescent images showing ATG9A and Vimentin signals in the recombinant grafts of different groups. Bar graph showing the numbers of ATG9A puncta per high power field in different groups. e, f Immunofluorescent images showing LC3 and Vimentin signals in recombinant grafts of different groups. Bar graph showing the numbers of LC3 puncta per high power field in different groups. g, h Immunofluorescent images showing Ki-67 and Vimentin signals in recombinant grafts of different groups. Bar graph showing proportions of Ki-67 positive cells in different groups. Scale bar, 50 µm in low power images or 10 µm in high-power images. **P < 0.01
Fig 3: Increased levels of key autophagy proteins in AppNL–G–F mice. Western blot analysis of cytosolic fraction of cortical brain homogenates from 12-month-old AppNL–F and AppNL–G–F mice were analyzed for Ulk1, p-Ulk1 S555 and p-Ulk1 S757 (A–E), Atg9A, Atg7, Atg5-Atg12, Atg16L (F–J), and the levels were quantified by densitometry (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001). Data are represented as mean ± SEM. Ns, not significant.
Fig 4: mATG9 and ATG16L1 Meet in Recycling Endosomes(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).(C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C.(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ***p < 0.001 and **p < 0.01.(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders’ coefficient). Error bar, SEM. NS, not significant; **p < 0.01.(H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ***p < 0.001 and **p < 0.01.(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.(J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ***p<0.001.(K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson’s coefficient). Error bar, SEM. ***p < 0.001.(L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson’s coefficient). Error bar, SEM. ***p < 0.001.(M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson’s coefficient). Error bar, SEM. ***p < 0.001.(N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson’s coefficient). Error bar, SEM. ***p < 0.001.See also Figure S3 and Movies S3 and S4.
Fig 5: SAP-enriched primary lysosomes are accumulated in the 1st layer of DNs. Fixed brain sections from 6-month-old APPNL-G-F mice were co-labelled with antibodies specific to LAMP1 and LAMP2 (A), LAMP1 and SAPs (B), LAMP1 and SAP-C (C), SAPs and SAP-C (D), LAMP1 and ATG9A (E), ATG9A and SAP-C (F), LAMP1 and RTN3 (G), SAP-C and RAB7 (H), and LAMP1 and ubiquitin (I). LAMP1 and SAP-C were largely co-localized with each other in the 1st layer of DNs, which was labeled by ATG9A, and surrounded by a 2nd layer marker, RTN3, and the 3rd layer markers RAB7 and ubiquitin. Scale bar is shown at the bottom right
Supplier Page from Abcam for Anti-ATG9A antibody [EPR2450(2)]