1. Oxidative Stress–Induced Calreticulin Expression and Translocation: New Insights into the Destruction of Melanocytes 
Yajun Zhang, Ling Liu, Liang Jin, Xiuli Yi, Erle Dang, Yang Yang, Chunying Li, Tianwen Gao 
Journal of Investigative Dermatology 
Volume 134, Issue 1, Pages (January 2014)
DOI: /jid
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2. Figure 1
Calreticulin (CRT) expression increased after exposure to hydrogen peroxide (H2O2)–induced oxidative stress. After exposure to specific concentrations of H2O2, CRT expression was evaluated in PIG1 and PIG3V cells at 0 minutes, 15 minutes, 30 minutes, 1 hour, and 2 hours of H2O2 treatment. (a, b) Cells were stained with annexin V and propidium iodide (PI) for 24 hours and analyzed by flow cytometry (FCM). Specific concentrations of H2O2 were chosen according to the induction of a statistically significant increase in early apoptosis (annexin-V+/PI−), before necrosis (annexin-V−/PI+ and annexin-V+/PI+; apoptosis rates: PIG1, 66.6%; PIG3V, 47.7%). (c, d, e, and f) CRT mRNA levels increased within 2 hours in both PIG1 and PIG3V cells. Total CRT protein levels showed similar results. Con, Control. One-way analysis of variance (ANOVA), followed by Kruskal–Wallis test, and followed by Dunn’s multiple comparison test were performed, yielding a P-value for all comparisons. *P<0.05 and ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Calreticulin (CRT) translocated to the surface of melanocytes under hydrogen peroxide (H2O2)–mediated oxidative stress. Surface externalized CRT (ecto-CRT) increased as the cells became apoptotic. (a) CRT was labeled with an antibody conjugated to Cy3 (red). Hoechst was used to stain the nucleus (blue) by immunofluorescence confocal microscopy (bar=60 μm). DAPI, 4',6-diamidino-2-phenylindole. (b, d) CRT increased in a time-dependent manner in both PIG1 and PIG3V cells, as observed by FACS. (c, e) CD47 expression decreased concomitant with an increase in CRT levels. One-way analysis of variance (ANOVA), followed by Kruskal–Wallis test, and followed by Dunn’s multiple comparison test were performed, yielding a P-value for all comparisons. *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Overexpression or knockdown of calreticulin (CRT) affected the way of melanocyte apoptosis. Upregulation of CRT caused increased hydrogen peroxide (H2O2)–induced apoptosis as compared with controls. Knockdown of CRT caused a reduction in H2O2-induced apoptosis as compared with controls. Transfected cells were cocultured with peripheral blood mononuclear cells (PBMCs) after pretreatment with or without H2O2 for 2 hours. ELISAs were used to measure the amounts of tumor necrosis factor-α (TNF-α) and IL-6. (a, b) Transfection with pCMV6-AC-CRT increased the susceptibility of melanocytes to apoptosis, whereas suppression of CRT expression decreased the rate of apoptosis, as measured by annexin-V/propidium iodide (PI) analysis. (c–n) Data are shown as differential cytokine production in cells cultured with or without H2O2. Each connected symbol represents paired samples from one individual donor. Paired t-tests were performed to evaluate the difference in cytokine production in cells cultured with or without H2O2. Two-way analysis of variance (ANOVA) was performed yielding a P-value for comparison between CRT siRNAs and scramble short interfering RNAs (siRNAs). *P<0.05; **P<0.005 and ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Effects of calreticulin (CRT) on the disease model in vivo. (a) Plasma CRT levels relative to lesion area in vitiligo patients, as measured by Spearman’s correlation. (b) CRT expression in the active and stable phases of vitiligo, as measured by ELISA. Each symbol represents one individual donor. Horizontal bars represent means or medians. (c, d) For detection of CRT and anti-CRT-specific IgG (n=25), the results are expressed as the difference in CRT concentration between healthy subjects and vitiligo patients (pg ml−1). Mann–Whitney U-test was performed. *P<0.05, **P<0.005, and ***P< (e) The role of CRT in physical and reactive oxygen species (ROS)–induced apoptosis in melanocytes. DC, dendritic cell; TNF-α, tumor necrosis factor-α.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions