1 Figure S1. XBP1 and EIF2AK3 in CoPs-induced autophagy in osteoblasts. ( A )
2 Western blots were performed after osteoblast cells were incubated with CoPs (50
3
µg/ml) for 12 h. (
B ) The density of the western blots bands presented in ( A ) was
4 quantified using ImageJ software. ( C ) Western blots performed after osteoblast cells
5 were incubated with siCtrl or siEif2ak3 before being stimulated with CoPs (50 µg/ml)
6 for 12 h. ( D ) The density of the western blots bands presented in ( C ) was quantified
7 using ImageJ software. ( E ) Immunofluorescence was performed after osteoblast cells
8 were incubated with siCtrl or siEif2ak3 before being stimulated with CoPs (50 µg/ml)
9 for 12 h. Scale bar: 10 µm. Red, LC3; blue, DAPI nuclear staining. ( F ) Quantification
10 of the percentage of cells with LC3 puncta presented in ( E ). siCtrl , siControl . Data are
11 presented as the means ± S.E.M. from 3 independent experiments. ** P < 0.01 versus
12 control.
13
14 Figure S2. Autophagy was involved in the toxicity of CoPs in osteoblasts. ( A ) CCK8
15 assay of osteoblasts treated with various concentration (0, 10, 50, 100 and 200 µg/ml)
16 of CoPs for 24 h. ( B ) CCK8 assay of osteoblasts cultured with various concentrations
17 (0, 0.1, 1 and 10 mM) of 3-MA prior to treatment with 50 µg/ml of CoPs for 24 h. ( C )
18 CCK8 assay of osteoblasts cultured with various concentrations (0, 0.1, 1 and 10 mM)
19 of 3-MA for 24 h. **, P < 0.01 versus control; ##, P <0.01 versus CoPs (50) group;
20 n.s., no significance.
21
22 Figure S3. Osteoblast senescence was not affected by CoPs in vitro. ( A ) SA-
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23 GLB1/SA-β-gal staining was measured after osteoblast cells treated with various
24 concentration (0, 10, 50, 100 and 200 μg/ml) of CoPs for 24 h. Representative images
25 are presented. ( B ) SA-GLB1/SA-β-gal staining was measured after osteoblast cells
26 were cultured with various concentrations (0, 0.1, 1 and 10 mM) of 3-MA prior to
27 treatment with 50 µg/ml of CoPs for 24 h. Representative images are presented. (
C )
28 Quantification of SA-GLB1/SA-β-gal-positive cells in ( A ). ( D ) Quantification of SA-
29 GLB1/SA-β-gal-positive cells in ( B ). n.s., no significance.
30
31 Figure S4. 3-MA decreased the expression of LC3 in osteoblasts in vivo.
32 Immunofluorescence was performed to determine the expression of LC3 in
33 osteoblasts. Sections of murine calvaria are presented for the animals from each
34 group. Scale bar: 50 µm. Red, LC3; green, osteoblasts (BGLAP); blue, DAPI nuclear
35 staining.
36
37 Figure S5. The effect of 3-MA on osteoclast formation in vivo. TRAP staining was
38 performed to identify osteoclasts. Sections of murine calvaria are presented for the
39
40 animals from each group. Osteoblasts are indicated by arrows. Scale bar: 200 µm.
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