Cadmium nanoparticles citrullinate cytokeratins within lung epithelial cells: cadmium as a potential cause of citrullination in chronic obstructive pulmonary disease

Figures & data

Figure 1 Detection of protein citrullination by Western immunoblot analysis.

Notes: A549 cells were left untreated (NC) or exposed to sub-toxic concentrations of Cd compounds. After 24 h of treatment, protein citrullination was quantified by Western blotting. A representative blot is shown, with actin used for loading control (A). The quantification analysis of the same blot is shown. The experiment was performed twice with comparable results (B). Cd, cadmium; NC, no treatment; CdO, treatment with CdO alone; Pre-CdO, exposure to CdO + ufCB pre-combustion; Post-CdO, exposure to CdO + ufCB post-combustion; Post-CdCl₂, exposure to cadmium chloride + ufCB post-combustion; ufCB, treatment with ultrafine carbon black alone.

Abbreviations: NC, negative control; CdO, cadmium oxide; CdCl₂, cadmium chloridel; ufCB, ultrafine carbon black.

Figure 2 Immunoprecipitation of citrullinated proteins for proteomic analysis.

Notes: A549 cells were left untreated (Ctrl), exposed to CdCl₂/ufCB post-combustion (Post-CdCl₂) or ufCB for 24 h and lysed. Cell lysates were immunoprecipitated, resolved by SDS-PAGE and visualized by Simply Blue™ SafeStain. Bands of interest, as indicated in the figure by arrows I, II and III, were excised and processed for nano-liquid chromatography-electrospray ionization.

Abbreviations: Ctrl, control; CdCl₂, cadmium chloridel; ufCB, ultrafine carbon black.

Figure 3 Immunofluorescent detection of protein citrullination in human lung epithelial cells A549.

Notes: NC, unexposed cells; CdO, cells exposed to nanoparticulate cadmium material; ufCB, cells exposed to ultrafine carbon black. Upper left panels, nuclei (blue); upper right panels, actin cytoskeleton (red); lower left panels, immunofluorescent staining for the presence of citrullinated proteins (green); lower right, merged staining panels. Scale bar, 10 μm.
Abbreviations: NC, negative control; CdO, cadmium oxide; ufCB, ultrafine carbon black.

Figure S1 A549 cell lysates probed for citrullinated proteins after treatment with Cd-containing materials.

Notes: Lanes, left to right, 1, no treatment; 2, CdCl₂; 3, CdO; 4, pre-combustion CdCl₂ + ufCB; 5, pre-combustion CdO + ufCB; 6, post-combustion CdCl₂ + ufCB; 7, post-combustion CdO + ufCB; 8, ufCB; 9, CdTe quantum dots.

Abbreviations: Cd, cadmium; CdO, cadmium oxide; CdCl₂, cadmium chloridel; ufCB, ultrafine carbon black.

Table S1 Identified citrullinated proteins with MS analysis scores over 100 in the immunoprecipitates of A549 cells after exposure to cadmium chloride (CdCl₂; post-combustion) and ultrafine carbon black (ufCB)

Accession	Description	Score	Coverage	# Proteins	# Unique peptides	# Peptides	# PSMs	# AAs	MW (kDa)	calc. pI
Post-combustion CdCl2 + ufCB
H6VRG0	Keratin 1	1,888.24	70.50	4	56	62	646	644	65.9	7.80
P35527	Keratin, type I cytoskeletal 9	1,812.51	80.58	1	40	40	545	623	62.0	5.24
P13645	Keratin, type I cytoskeletal 10	519.10	58.22	1	27	32	168	584	58.8	5.21
P35908	Keratin, type II cytoskeletal 2 epidermal	343.55	61.35	1	29	41	115	639	65.4	8.00
P04259	Keratin, type II cytoskeletal 6B	304.03	54.26	1	2	32	96	564	60.0	8.00
Q0IIN1	Keratin 77	228.62	14.36	2	5	9	73	578	61.8	5.85
B4DRR0	cDNA FLJ53910, highly similar to Keratin, type II cytoskeletal 6A	213.23	62.80	2	4	36	73	535	57.8	8.00
P13647	Keratin, type II cytoskeletal 5	189.16	53.56	1	23	36	63	590	62.3	7.74
P02533	Keratin, type I cytoskeletal 14	113.20	51.69	1	6	24	36	472	51.5	5.16
P08779	Keratin, type I cytoskeletal 16	111.65	46.72	1	8	22	35	473	51.2	5.05
ufCB
H6VRF8	Keratin 1	2,432.14	77.33	3	59	66	813	644	66.0	8.12
P35527	Keratin, type I cytoskeletal 9	2,225.33	82.50	1	48	48	627	623	62.0	5.24
P13645	Keratin, type 1 cytoskeletal 10	778.05	65.07	1	31	37	243	584	58.8	5.21
P35908	Keratin, type II cytoskeletal 2 epidermal	521.35	73.24	1	37	47	177	639	65.4	8.00
P04259	Keratin, type II cytoskeletal 6B	287.78	53.72	1	2	32	93	564	60.0	8.00
Q0IIN1	Keratin 77	208.62	19.03	2	8	13	64	578	61.8	5.85
P13647	Keratin, type II cytoskeletal 5	203.56	53.90	1	26	38	69	590	62.3	7.74
B4DRR0	cDNA FLJ53910, highly similar to Keratin, type II cytoskeletal 6A	188.92	55.89	3	4	32	64	535	57.8	8.00
P02533	Keratin, type I cytoskeletal 14	112.72	41.95	1	8	20	36	472	51.5	5.16
P08779	Keratin, type I cytoskeletal 16	108.85	42.28	1	7	20	37	473	51.2	5.05
B4DRW1	cDNA FLJ55805, highly similar to Keratin, type II cytoskeletal 4	101.73	20.46	4	6	10	32	474	51.7	6.81

Abbreviations: MS, mass spectrometry; CdCl₂, cadmium chloridel; ufCB, ultrafine carbon black; PSM, peptide spectrum matches; AA, amino acids; calc. pl, calculated isoelectric point.