C. Wu, J. Zheng, X. Yao, H. Shan, Y. Li, P. Xu, X. Guo

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Defective autophagy in chondrocytes with Kashin-Beck disease but higher than osteoarthritis C. Wu, J. Zheng, X. Yao, H. Shan, Y. Li, P. Xu, X. Guo Osteoarthritis and Cartilage Volume 22, Issue 11, Pages 1936-1946 (November 2014) DOI: 10.1016/j.joca.2014.08.010 Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Chondrocyte morphology under TEM observation (A–C, TEM × 3,000; D–F, TEM × 10,000). Chondrocytes in normal cartilage showed normal nuclear and cytoplasmic morphology such as abundant and compact mitochondria, and slightly dilated ER (A and D). KBD chondrocytes showed deformed nuclear, swelled mitochondria, and a great number of lysosomes (B). Gross microfilaments were pervasive around giant nuclei, and masses of glycogen flooding the cytoplasm (E). The main characteristics of OA chondrocytes were severely dilated ER and several degenerating lysosomes and unrecognizable organelles (C and F). Osteoarthritis and Cartilage 2014 22, 1936-1946DOI: (10.1016/j.joca.2014.08.010) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Immunohistochemical localization of Beclin1 (A) and LC3 (B) in cartilage. n = 6 in each group. In the bars, values are the mean and SD. * = P ＜ 0.05, & = P ＜ 0.01. (A and B), a–c, original magnification ×200 of superficial zone (SZ) and middle zone (MZ) of normal, KBD and OA cartilage. d–f, original magnification ×400 of middle zone. g–i, original magnification ×400 of deep zone (DZ). Beclin1, P = 0.034 SZ of normal vs SZ of KBD; P = 0.001 DZ of normal vs DZ of KBD. LC3, P = 0.032 SZ of normal vs SZ of KBD; P = 0.002 SZ of KBD vs SZ of OA; P = 0.116 MZ of normal vs MZ of KBD. The others P values < 0.001. Osteoarthritis and Cartilage 2014 22, 1936-1946DOI: (10.1016/j.joca.2014.08.010) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Immunoblotting of cellular Beclin1, LC3, LC3-II and p62. The representative immunoblotting images of Beclin1, LC3 and p62 and the bar of densitometric analysis. n = 6 in each group.* = P ＜ 0.05, & = P ＜ 0.01. Beclin1, P = 0.039 Normal vs KBD; P = 0.040 KBD vs OA. LC3, P = 0.010 Normal vs KBD; P = 0.007 KBD vs OA. LC3-II, P = 0.710 Normal vs KBD; P = 0.010 Normal vs OA; P = 0.020 KBD vs OA. p62, P = 0.430 Normal vs KBD; P = 0.134 Normal vs OA; P = 0.030 KBD vs OA. The others P values < 0.001. Osteoarthritis and Cartilage 2014 22, 1936-1946DOI: (10.1016/j.joca.2014.08.010) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 The microarrays data analysis. (A) The fluidogram of microarrays data analysis. (B) The dimension of two comparisons KBD vs normal and KBD vs OA. * means increased expression when KBD vs Normal, and decreased expression when KBD vs OA; # means decreased expression when KBD vs Normal, and increased expression when KBD vs OA; no sign means increased expression when KBD vs Normal, and increased expression when KBD vs OA. (C) The network of significant genes in two comparisons analyzed by IPA. (D) Histogram showing levels of expression of five selected genes, as measured by microarray (dark gray bars: n = 4) and RT-PCR (light gray bars: n = 6). Values are the mean ± SD. *=P < 0.05, & = P < 0.01. a, KBD vs the normal. The P values of BCL2, BNIP3, BNIP3L, BAX, BAK1 were 0.045, 0.044, 0.001, 0.006, and 0.034, respectively. Ratio > 0 means gene expression level was higher in KBD. b, KBD vs OA. The P values of BCL2, BNIP3, BNIP3L, BAX, BAK1 were 0.022, 0.114, 0.035, 0.027, and 0.032, respectively. Ratio > 0 means gene expression level was higher in KBD, and Ratio < 0 means gene expression level was higher in OA. Osteoarthritis and Cartilage 2014 22, 1936-1946DOI: (10.1016/j.joca.2014.08.010) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Measurements of autophagy and cell death. n = 6 in each group. (A) Fluorescence measurements of autophagic level (MDC fluorescence) and cell death (PI fluorescence). a, The image of MDC and PI fluorescence stain. MDC, emitting in blue; PI, emitting in red. b, the scatterplot of fluorescence intensity. Each plot presents one sample. c, the bar graph of fluorescence intensity ratio. The ratio is fluorescence intensity of MDC to PI. P < 0.001 normal vs KBD, normal vs OA and KBD vs OA. (B) Chondrocyte apoptosis was measured by FCM. The three detected samples above were the normal, KBD and OA chondrocytes, respectively, and the three below were corresponding chondrocytes with rapamycin treatment. Osteoarthritis and Cartilage 2014 22, 1936-1946DOI: (10.1016/j.joca.2014.08.010) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Indicators of autophagy and mitochondrial function. n = 6 in each group. (A) a, Co-localization of autophagic vacuoles (MDC blue fluorescence) with mitochondria (MitoTracker Red fluorescence). b, the scatterplot of fluorescence intensity. Each plot presents one sample. c, the bar graph of fluorescence intensity ratio. The ratio is fluorescence intensity of MDC to MitoTracker. P = 0.005 normal vs KBD; P = 0.025 normal vs OA; P = 0.409 KBD vs OA. (B) Double staining of autophagy (MDC blue fluorescence) and ROS (DCF green fluorescence). b, the scatterplot of fluorescence intensity. Each plot presents one sample. c, the bar graph of fluorescence intensity ratio. The ratio is fluorescence intensity of MDC to DCF. P < 0.001 normal vs KBD; P < 0.001 normal vs OA; P = 0.253 KBD vs OA. Osteoarthritis and Cartilage 2014 22, 1936-1946DOI: (10.1016/j.joca.2014.08.010) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions