Image studio software version 5

Cells were washed twice with ice-cold phosphate-buffered saline, before being lysed with the addition of ice-cold lysis buffer. Frozen heart tissue was crushed and lysed in ice-cold lysis buffer. Protein (9 μg for cells and 30 μg for tissue) was loaded onto SDS-PAGE gels and separated by electrophoresis. Even protein loading and transfer were confirmed by either the loading control β-actin or Ponceau S staining. Bands were quantified using LI-COR C-Digit chemiluminescent detection system (LI-COR Biotechnology, Lincoln, Nebraska) and Image Studio software version 5.2.5 (LI-COR).

ER-enriched fractions from BV2 microglia cells were prepared using a microsome isolation kit (BioVision, Milpitas, CA) according to the manufacturer's instructions. Western blot analysis was performed as previously described [10 (link)]. Proteins were separated by 15% SDS-PAGE and transferred to nitrocellulose. Transferred slabs were stained in Coomassie blue to verify equal loading between samples. Membranes were incubated in a CXorf56 antibody (1 : 500) or a calnexin antibody (1 : 100) overnight at 4°C, and primary antibodies were visualized using the goat anti-rabbit HRP-linked secondary antibody, incubated for 1 hour at room temperature (1 : 5,000) (Jackson's Laboratory, West Grove, PA), followed by ECL detection. To confirm equal protein loading, Western blot analysis was also carried out using a beta-actin antibody at 1 : 50,000). Densitometry analysis was performed using the Image Studio software, version 5.2.5 (LI-COR Biosciences).

Quantitative analysis was carried out using ImageStudio Software Version 5.2.5 (LI-COR Biosciences) on non-saturated Western blot bands. The signal intensity of each Western blot band was collected using the Western blot analysis setting on ImageStudio that subtracts the background signal of each lane from the signal of each band of interest. Band signal intensity values were not normalized to a loading control because the values calculated for quantitative analysis were ratios that corresponded to the signal intensity of an individual lane’s cleavage fragment band divided by the signal intensity of the total N-terminal signal recorded for both the full-length protein and corresponding N-terminal fragment in each lane. Note that while for some blots we adjusted brightness and contrast, this had no impact on quantification. Indeed, signal quantification is based on the original Western blot image capture (see above), and is thus independent of subsequent brightness and contrast adjustments.
Statistical analysis was performed with Prism 9 (GraphPad). Unpaired t-test was used to determine statistical significance when comparing two sets of measurements. One-way ANOVA followed by Dunnett’s multiple comparisons test was used for experiments with more than two groups. All error bars in graphs represent the standard error of the mean.

To measure the extracellular signal-regulated protein kinase (ERK1/2), thyrospheres were lysed and subjected to Western blot analysis, as previously described (20 (link)). The following antibodies against total and phosphorylated ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-ERK1/2, anti-P-ERK1/2 (T202/Y204) and anti-Vinculin. Bands were detected digitally using the Odyssey Fe imaging system (Li-COR Bioscience, Lincoln, NE, USA) and the blots were then quantified using the Li-COR Image Studio software version 5.2.5.
The metal concentration used for evaluating ERK1/2 activation at 5, 15 and 30 minute time points was selected on the basis of the peak effect of each metal on BrdU incorporation.
ERK phosphorylation was also measured in cells exposed to metals in the presence of PD98059 (20 μM) that was kept in the culture medium throughout the entire period of exposure to metals.

A total of 180 spheroids were collected and dissociated as described before. Samples were washed and lysed with 1x RIPA buffer (Bioconcept, Allschwil, Switzerland, CellSignaling, 9806S) supplemented with phosphatase inhibitor (Roche, Basel, Switzerland, 04906837001) and protease inhibitor (Roche, 11836170001) for ≥10 min on ice. The extract was centrifuged for 10 min at 14,000 rpm at 4 °C. The protein concentration was determined using the Bradford reagent (Sigma-Aldrich, B6916-500ML) and by analyzing the absorbance at 595 nm on a Biotek Cytation 3.
A quantity of 50 µg of total protein content was loaded on SDS-page gels (Thermofisher, Invitrogen, NP0321BOX). Proteins were separated based on their molecular weight through gel electrophoresis. The gel content was transferred onto a nitrocellulose membrane (Amersham Protran 10600007) and protein bands were identified through immunofluorescence staining (Table S5). The analysis was done on the LI-COR scanner and the intensity was analyzed using the ImageStudio software version 5.2 (LI-COR Biosciences, NE, USA).