Gel doc apparatus

The extraction of total RNA was performed using TRIZOL (Life Techonologies, USA) by following the suggested protocol. RNA reverse transcription into cDNA was carried out by the SuperScriptTM II Reverse Transcriptase (Life-Techonologies) using random hexamer primers. cDNA amplification was achieved by using PCR Master Mix 2X (Fermentas-Dasit, Italy) and specific primers for human MAP-2, NeuroD1, GCLC, GCLM, HO-1, Nrf2, Bach1 and GAPDH. All the primer sequences used have been synthesized at Tib Mol Biol, Italy and listed in supplementary table 1. After separation on 2% agarose gel, a densitometric analysis of PCR products, stained with ethidium bromide and visualized under UV light, has been performed using a GelDoc apparatus (Bio-Rad, Italy). The expression of all the genes analyzed have been normalized on the expression of GAPDH.

Intact follicles were harvested at appropriate time intervals, washed (x 3), and lysed in ice-cold oocyte extraction buffer as described earlier (Biswas and Maitra, 2021 (link)), supernatant separated by spinning at 17,500×g for 20 min at 4°C, and stored at 80°C until further use. Following the quantitation of protein concentration, oocyte lysates (50 µg/lane) from each treatment group were resolved through SDS-PAGE and immunoblotted following our standard laboratory protocols (Das et al., 2013 (link); Maitra et al., 2014 (link)). The electroblotted Hybond-P PVDF membrane was blocked in 5% BSA in TBST (pH 7.6, Tris 50 mM, NaCl 150 mM, Tween-20 0.1%), incubated with primary antibody (1:1000) overnight at 4°C followed by secondary (1:2000) antibody incubation for 2 h at RT (for details see Supplementary Table S1). BCIP-NBT was used as the substrate to develop the bands, visualized, and recorded in Gel Doc apparatus (Bio-Rad). The signal intensity of target bands in each lane was measured through ImageJ software and neutralized by endogenous factor (anti-actin or respective total protein) for that lane. The normalized target signal for each sample is then divided by the normalized target signal observed in the experimental control sample to generate the fold change in target protein (n = 3 technical replicates; n = 3 biological replicates) (Pillai-Kastoori et al., 2020 (link)).

The DNA restriction fragments were separated by electrophoresis in a 3 % (wt/vol) high-resolution agarose (Prona) gel with ethidium bromide (0.5 mg/mL) in 0.5x Tris-borate-EDTA (pH 8.0) buffer at 90 V for 60 min and visualized under a UV source. Each gel was documented with a GelDoc apparatus (BioRad, USA). For all investigated bacterial strains (reference and wild), restriction fragment sizes were measured (in bp) by comparison with the M100-1000 bp DNA Ladder (Blirt, Poland) using Quantity One software (BioRad).

Gelatin zymography was performed as previously described^{36 (link)}. SDS-PAGE gels (7.5%) were prepared by incorporating 0.1% gelatin during acrylamide/bis-acrylamide polymerization. In all lanes, 10 µl of the supernatant, obtained from cultured hippocampi, was loaded. Gels were subjected to electrophoresis at 125 V constant. After electrophoresis, SDS was removed by washing gels for 30 min with a renaturating solution containing (in mM): 50 Tris-HCl, pH 7.4, and 2.5% Triton X-100. Gels were further washed with large volumes of ddH₂O to remove residual Triton X-100. To assess MMPs activity gels were incubated overnight at 37 °C in an activation buffer containing (in mM): 50 Tris-HCI, pH 7.4, 120 NaCl, 5 CaCl₂. Accordingly to the experimental setting, activation buffer was supplemented with CQ (10 µM), TPEN (100 µM), or vehicle (DMSO 1%). Gels were then incubated for 30 min with a developing solution containing (in %): 0.1 Coomassie blue, 40 methanol, 10 acetic acid, 50 ddH₂O. Staining was stopped when clear gelatinolytic bands appeared. Gels were photographed with a Gel-Doc apparatus (Biorad) and bands quantified by densitometry by using the Fiji distribution of ImageJ (v 1.51)^{92 (link)}.