Additional file 1: Table S1. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

Particle physicochemical properties and exposure information. (XLSX 10 kb

Additional file 4: Table S3. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

Details of rodent studies considered by NIOSH in calculating points of departure (PODs). (DOCX 15 kb

Additional file 3: Figure S1. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

Heatmap showing the dose at which each pathway was significantly perturbed. Each column represents a post-exposure time point for the denoted MWCNT, and each row represents a signifncatly perturbed pathway. All colored cells represent the lowest dose at which the pathway is perturbed. Blank cells represent pathways that were not significantly perturbed. (PPTX 76 kb

Additional file 2: Table S2. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

List of all significant pathways associated with the molecular initiating event (MIE), each key event (KE), and each associative event (AE) including the benchmark dose (BMD) value (BMDL values included in parentheses) for each MWCNT investigated at each time-point. n.a. indicates the BMD(L) could not be calculated due to poor model fit. (XLSX 18 kb

Ubc9 co-localizes with wild-type and mutant lamin A/C.

<p>Confocal microscopy images of nuclei of C2C12 cells expressing wild-type and mutant lamin A-CFP, DsRed2-lamin C and ubc9-GFP. The lamin constructs used as well as the associated pathology are indicated at the top. Lamin A and lamin C images are represented in one panel as they co-localize in all cells. Cells were visualized by wide-field fluorescence microscopy with excitation wavelengths of 434 nm for lamin A-CFP, 558 nm for DsRed2-lamin C, and 588 nm for ubc9-GFP. Each picture presented is representative of the most commonly observed phenotype.</p

Lamin A and C are not sumoylated by sumo1.

<p>(A–C) Western blots of C2C12 nuclear proteins: (A) Untransfected (UNT) or sumo1-YFP transfected, or triple transfected wild-type lamin A-CFP, DsRed2-lamin C and sumo1-YFP probed for lamin A/C. NEM was included with harvesting of selected samples to stabilize sumo conjugation. (B) Western blot A stripped and reprobed for the reversibly sumoylated protein, SP3. (C) UNT or triple transfected wild-type and mutant DsRed2-lamin C, wild-type sumo1-YFP and wild-type Ubc9-HA probed for lamin A/C. (D–E) Immunoprecipitation of endogenous and exogenous lamin A and C. (D) Nuclear extracts of sumo1-HA transfected C2C12 cells were immunoprecipitated using anti-lamin A/C antibody and western blotting was performed for lamin A/C. (E) Nuclear extracts of lamin A-CFP, lamin C-CFP, or empty CFP vector transfected COS7 cells were immunoprecipitated using anti-GFP tag antibody and western blotting was performed for the GFP tags. All protein was harvested in the presence of NEM. Emerin, a known lamin A/C binding partner, was included as a positive control for immunoprecipitation.</p

Sumo1 localization is disrupted in soleus muscle sections from <i>Lmna</i>^H222P mice.

<p>Fluorescent microscopy images of soleus muscle cross sections from <i>Lmna</i>^+/+ and <i>Lmna</i>^H222P/H222P mice. Top row sections were stained for sumo1 (green). Bottom row sections were stained with only secondary antibody to show background signal of anti-mouse-568 nm antibody (green). All sections were counterstained for DAPI (blue). Each picture presented is representative of the most commonly observed phenotype. Scale bar represents 10 µm. White arrow highlights one cell with nuclear aggregation of sumo1.</p

Sumo1 localization is disturbed by endogenous p.<i>His222Pro</i> mutant lamin A/C in primary mouse myoblasts.

<p>Fluorescent microscopy images of non-transfected and sumo1-YFP transfected <i>Lmna</i>^+/+ (WT) and <i>Lmna</i>^H222P/H222P primary myoblast nuclei. (A) Untransfected myoblasts immunostained for endogenous sumo-1 (green). (B) Sumo1-YFP transfected myoblasts expressing YFP-tagged sumo1 (green). All myoblasts were counterstained for DAPI (blue). Each picture presented is representative of the most commonly observed phenotype.</p

Quantitative analysis of colocalization of sumo1 and mutant lamin A and C.

<p>Quantitative colocalization analysis of sumo1 and lamin A and C was performed using the ImageJ- 1.46r software. Thresholded Manders coefficients tM1 and tM2 and the percentage of pixels intensity above threshold colocalized (% Ch1 or Ch2 int > thresh) in each of the two channels (Ch1 for sumo (green fluorescence) and Ch2 for lamin A and C (red fluorescence) were calculated using the threshold algorithm of Costes et al (2004) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045918#pone.0045918-Costes1" target="_blank">[43]</a>.</p

Mutant lamin A/C expression increases levels of sumoylated proteins.

<p>Western blot analysis of sumo1 in nuclear protein harvested from C2C12 untransfected (UNT) cells or transfected with wild-type (WT) or mutant lamin C-CFP and sumo1-YFP harvested with NEM. Blots were re-probed for GapDH as a loading control.</p