Fig 2: Specific silencing on mutant allele and protein is maintained over time(A) Semi-quantitative Dnm2 and Atpase RT-PCR products and quantification of Dnm2 expression normalized to Atpase (HTZ n ≥ 10; WT n = 12). (B) EcoNI digestion profile of Dnm2 PCR products and quantification of the mutant/WT, mutant/Atpase, and WT/Atpase ratios (HTZ and WT n = 11). (C) DNM2 western blot and quantification of signal by densitometry. GAPDH was used as loading control (HTZ and WT n = 8). In scatterplots (A–C), the bars are mean values and error bars indicate SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.1 two-tailed using a Mann-Whitney U test comparison. HTZ, heterozygous; Mut, mutant; WT, wild type.

Fig 3: High dose of therapeutic AAV-sh9 does not improve the benefit in old mice(A) Muscle mass relative to body weight in milligrams and grams, absolute maximal force in grams, and specific maximal force (in grams/mg) developed by TAs from WT and AAV-injected HTZ mice (WT n = 8, HTZ-control n = 7, and HTZ-sh9 n = 4). (B) Quantification of Dnm2 expression normalized to Atpase from semi-quantitative Dnm2 and Atpase RT-PCR (left) and quantification of the mutant/WT ratio from EcoNI digestion profile of Dnm2 PCR (right; WT ≥ 4, HTZ-Ctl n = 4, and HTZ-sh9 = 4). (C) Quantification of the vgs per nanogram of DNA in HTZ 6-month-old mice injected with AAV1-sh9 at low (n = 4) or high dose (n = 4). (D) Representative H&E staining of TA sections from WT and AAV-injected HTZ mice. Scale bars represent 50 μm. In histogram and scatterplots (A–C), bars represent mean ± SEM. Statistical analysis was performed using Mann-Whitney U test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 4: One to two dozen ITSN1 molecules are recruited to each CME site(A and B) 2D histograms of the first two PCs forAP2 and ITSN1 (A) and AP2 and DNM2 (B). Shown are dynamic features extracted from live-cell imaging data collected on the same days using the same imaging parameters. Tracking and plotting methods are as described in Figure 2A. The figures show the numbers and percentiles of tracks in each individual cluster.(C) Averaged intensity vs. time plots for ITSN1 and DNM2 in standard and slow CME events (cluster 4, lifetime ≥ 30 s with ≥ 5 consecutive frames of ITSN1 or DNM2 detections). Tracks are aligned at the ITSN1 or DNM2 peak (time 0). Note that AP2 intensities are not shown in the plot. Data are represented as mean ± ¼ SD. The number of tracks, along with the average and standard deviation of the maximum intensities of ITSN1 and DNM2, are displayed.(D) Cumulative histogram of the fraction of ITSN1- or DNM2-positive standard and slow CME tracks (cluster 4, lifetime ≥ 30s with ≥ 5 consecutive frames of ITSN1 or DNM2 detections) at a given time after the AP2 signal is first detected (time 0). n = 2,307, 2,394.(E) Averaged intensity vs. time plots for different event types of cluster 4 tracks. Tracks with equal to or more than 5 consecutive frames of ITSN1 or DNM2 detections are grouped based on AP2 lifetimes and aligned at the first detection of AP2 (time 0). Numbers of tracks in each type of events are shown. Data are represented as mean ± ¼ SD.See also Figure S3.

Fig 5: Activated PKCα enhanced PEV production and promoted lung metastases in nude mice. (A) PEVs produced by platelets from tumor-bearing mice treated with PMA, GÖ6976, or DMSO, and from non-tumor-bearing mice were counted using small-particle flow cytometry, and are shown as the mean ± standard deviation for the number of microparticles per microliter. (n = 10) (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Western blotting analysis of PKCα and DNM2 expression in platelets from tumor-bearing mice treated with PMA, GÖ6976, or DMSO, or non-tumor-bearing mice treated with phosphate-buffered saline (PBS). (C) Representative images and semi-quantitative analysis of lung metastases in nude mice using luciferase-based bioluminescence imaging. (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Representative images and quantification of visible metastatic lung nodules in mice from the three groups (yellow arrows). (*p < 0.05, **p < 0.01, ***p < 0.001). (E) Hematoxylin and eosin (H&E) staining of lung metastasis nodes. Original magnification: 10×.