AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells

Abstract

An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/ p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

microRNA-17 regulates the expression of ATG7 and modulates the autophagy process, improving the sensitivity to temozolomide and low-dose ionizing radiation treatments in human glioblastoma cells

“…The miR-17-mediated activation of autophagy, through the formation of autophagosomes, was also scored in T98G living cells, transduced with the Premo Autophagy Sensor (LC3B-FP) BacMam 2.0 system and subsequently analyzed with the AUTOCOUNTER ImageJ Javascript tool. 35 As a result, T98G cells treated with miR-17 inhibitor scored an increase in the surface amount of punctate fluorescent vesicles, suggesting a LC3B-FP protein accumulation in nascent autophagosomes. 49 Next, autophagy induction due to miR-17 downregulation was assayed as an adjuvant strategy to current in vitro chemo-and radio-therapeutic schemes.…”

“…In addition, rapamycin administration increased LC3B-FP signals, further enforcing the punctuated pattern of LC3B-FP in AmiR-17 transfected cells. Statistics on vesicles area and numbers in T98G transfected cells, exposed to the above mentioned rapamycin doses and AmiR-17 or PmiR-17 transfection, were calculated using the AUTOCOUNTER ImageJ Javascript tool: 35 according to these analyses, AmiR-17 administration induced a significant increase in the autophagiclike vesicles areas, particularly within rapamycin-treated cells (P < 0.005), rather than a direct increase in their total number within the cells (Fig. 5).…”

“…46 In particular, human OncomiR-1 is ) and number of total vesicles (Ves tot ) are calculated using the aUTOCOUNTeR ImageJ Javascript tool as described. 35 Results are obtained analyzing different DIC phase contrast-fluorescent photographs for a total of 30 T98G cells for each of the mentioned mock, rapamycin, amiR-17, pmiR-17 treatments. *P < 0.005.…”

“…35 Briefly, the program analyzed DIC phase contrast-fluorescent photographs with operator-defined cells contours; then, the number of intracellular fluorescent spots (Ves tot ) and their areas percentage within the cell (A ves /A cell ) were calculated by an operator-independent algorithm. Results were obtained analyzing a total of 30 T98G cells for each of the mentioned mock, rapamycin, AmiR-17/PmiR-17 treatments.…”

Mutual exacerbation of peroxisome proliferator‐activated receptor γ coactivator 1α deregulation and α‐synuclein oligomerization

“…An inverted fluorescence microscope (Axio observer-A1, Zeiss, Göttingen, Germany) was employed for live cells imaging using 60× magnification. Quantification of autophagosome was performed using the plugging ImageJ autocounter (http://rbsweb.nih.gov/ij/) as described previously 60 .…”