mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.


a b s t r a c t
We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy Western-blotting analysis of LC3 and p62 proteins using Sample buffer (SB) 1X lysis buffer, detection of endogenous LC3 and p62 immunofluorescence and p62 mRNA levels by qPCR.

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Value of the data
This data provides characterization of basal macroautophagy flux and response to classical inducers (EBSS) and inhibitors (Baf. A1) of this mechanism in MEFs, HFs, SH-SY5Y and N27 cell lines.
The data would be valuable for further studies of autophagy dynamics in these four cell lines. This data could give a basis for further experiments on revealing the underlying mechanism of autophagy in these cell lines.
The data support the development of transcription analysis of autophagy markers.

Data
In order to understand autophagy dynamics [1] and develop a robust methodological assay, we used four cell models: MEFs, HFs, SH-SY5Y human neuroblastoma cells, and N27 rat mesencephalic dopaminergic cells. LC3 and p62 protein levels were analyzed by Western-blotting assay, using SB 1X as a lysis buffer (Fig. 1). Furthermore, we measured the endogenous immunostaining of LC3 and p62 proteins in all cell models used in this report (Fig. 2). To complete this data analysis, p62 mRNA expression was performed in all cell lines (Fig. 3).
After 24 h , the culture medium was replaced with different treatments (Control, EBSS, Baf. A1 and Baf. A1 þEBSS). To induce autophagy by starvation conditions, the culture medium was replaced with EBSS (Sigma-Aldrich, E2888). To block fusion between autophagosomes and lysosomes, cells were incubated with 100 nM Baf. A1 (LC Laboratories, B-1080). For combined treatment, cells were preincubated with 100 nM Baf. A1 for 1 h and then they were washed with phosphate-buffered saline (PBS) 1X and treated with 100 nM Baf. A1 and EBSS. All treatments lasted 4 h.

Western-blotting
Following treatments, cells were processed as described previously [2]. Basically, cells were washed with PBS 1X and lysed in sample buffer (SB) 1X (2% (v/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, and 50 mM Tris-HCl, pH 6.8, in distilled water) by pipetting until homogenization. Protein concentration was measured based on the bicinchoninic acid assay, using bovine serum albumin as a standard. Samples were heated at 95°C for 10 min before their quantification.

Immunofluorescence
For the detection of endogenous p62 and LC3B, cells were seeded on coverslips, fixed with paraformaldehyde (4% in PBS 1X) and permeabilized with Triton X-100 solution (0.1% in PBS 1X) for 10 min. To block non-specific binding, cells were incubated with 10% FBS in PBS 1X for 20 min, followed by incubation with primary antibodies anti-p62 (1:500) and anti-LC3B (1:500) for 1 h at room temperature. After that, cells were incubated with Alexa Fluor 488 anti-rabbit (1:1000) (Molecular Probes, A-11034) and 568 anti-mouse (1:1000) (Molecular Probes, A-11004) secondary antibodies for LC3 and p62, respectively. Finally, coverslips were mounted on microscope slides, by using fluoromount-G (SouthernBiotech, 0100-01) medium. Images were taken by using an inverted fluorescence microscope (Olympus, IX-51) equipped with a camera (Olympus, DP70). The quantitative measurement of the fluorescence signal was performed using ImageJ software analyzing at least 200 cells per condition. Immunofluorescence procedure was developed as previously described [2].

Quantitative PCR
Total RNA was extracted by RNeasy Mini Kit (Qiagen, 74104); 500 ng of total RNA were reversetranscribed into complementary DNA by using a QuantiTect Reverse Transcription Kit (Qiagen, 205311), both according to the manufacturer's protocol. p62 mRNA expression was measured by qPCR with KAPA SYBR Fast reagents (KK4601), by using the primers described above. GAPDH gene expression was used as an endogenous control, and the expression level was calculated by using the (2 -ΔΔCt ) ratio [4].

Statistical analyses
Each experiment was repeated at least three times. The data were analyzed by two-tailed unpaired Student's t-test and ANOVA test where applicable, and all comparisons with a p value less than 0.05 (p o0.05) were considered statistically significant (***po 0.001, **p o0.01, *p o0.05). Non-significant results are not indicated in the figures. The data are expressed as the mean 7the standard error of the mean (SEM).