Fig 1: Altered expression of autophagy-related proteins (ATGs) in Human uterine fibroid. RNA was extracted from frozen myometrium tissue and reverse transcribed as described in the Materials and Methods. (a) Real-time PCR analysis of mRNA levels of several ATGs. Data show significantly higher expression of ATG3, AtG7, ATG12, ATG16, but not ATG4, in HuLM compared to UTSM. (b–e) Quantification of the ATG4D isoforms by real-time PCR showing lower expression of ATG4D isoform. (f, g) Western blot analysis of ATG4D and the expression of ATG4D normalized to Actin. Data represent mean±SEM of three independent experiments. *P<0.05.
Fig 2: Initiation of autophagy is enhanced in Human UFs. Fibroid tissues (F) and adjacent normal myometrium (MyoF) collected from different patients, as well as in fibroid cell line (HuLM) and normal myometrial cells (UTSM) were analyzed for the expression of markers of autophagy flux by western blot. (a) Human UFs biopsy (MyoF, adjacent normal myometrium and F, UF). (b) Cell lines UTSM, HuLM. Levels of lysate proteins loaded in the gels were monitored by immunoblotting with an anti-Actin antibody. (c and d) the relative expression of LC3I/II, P62, ATG4D and Beclin1 was calculated based on the grayscale value of ß-actin from patient biopsies and UF human cell lines respectively. (e) Quantification of endogenous of LC3 and P62 by flow cytometry. Histograms show a higher expression of LC3 and p62 in HuLM compared to UTSM. Data are from one experiment and representative of three independent experiments. (f) Immunohistochemistry staining of LC3 A/B in (MyoF) and (f) from UF patients (40× magnification). (g) Immunofluorescence staining for LC3 in human fibroid cells (magnification 63×). Representative images are shown. Data are means of three independent experiments. *P<0.05, **P<0.01, ***P<0.001.
Fig 3: ATG4s are not induced in Human UFs patient biopsies and fibroid cell line. (a) Real-time PCR quantification of total ATG4 mRNA expression and ATG4 isoforms. (b) Immunohistochemistry staining with human anti-ATG4 and anti-ATG4D. (c) Immunofluorescence staining for ATG4D in UTSM and HuLM cell lines, Green ATG4D and Blue is DAPI. (n=15, *P< 0.05).
Fig 4: Knock-down of ATG4D by shRNA in UTSM fibroid cell line increase proliferation. The expression of ATG4D in UTSMs was knocked-down by ATG4D-specific shRNA using lentivirus particles. Control cells were infected with lentivirus containing non-specific shRNA vector. (a) Silencing the expression of ATG4D in UTSM by shRNA and scramble control both tagged with GFP construct. (b) Real-time PCR of ATG4D mRNA expression. (c) Western blot of ATG4D protein. (d) Proliferation assay analysis by FACS intracellular staining for Ki67. (e) The cells (3000/well) from either ATG4D knockdown or scrambled control were seeded onto 96-well tissue culture plates. The MTT assay was performed at different time points 24, 48, 72, and 96 h. The Averaged cell numbers from triplicate wells were used in preparing the showed data graph. Each data point is the mean (±S.D.) from an individual experiment performed in triplicate (n=3). *P<0.05, **P<0.01.
Fig 5: Knock-down of ATG4D by shRNA in UTSM cell line mimic UF phenotype. (a) Autophagy markers analysis by FACS with intracellular staining and mean fluorescence intensity MFI for LC3, and (b) by western blot for LC3 and P62. (c) Extracellular matrix markers analysis by western blot. (d) Endogenous quantification of pro-inflammatory cytokines expression was done by intracellular staining for TNF-a, IL-1ß, TGF-ß and IL-10 using flow cytometry analysis. The expression level was represented as mean fluorescence intensity (MFI). Data from the histogram are shown as mean±S.D. and are representative of three independent experiments. *P<0.05, **P<0.01, ***P<0.001.
Supplier Page from Abcam for Anti-ATG4D antibody