Figures & data
Figure 1. Brief summary of canonical calreticulin (CRT) exposure pathway.
Calreticulin exposure is the marker of ICD and its canonical mechanism is widely described with three sequential modules: ER stress, apoptotic and translocation. ER stress module: ER stress is the initiating cause of ICD by all ICD inducers. ER stress starts with the phosphorylation of eIF2α kinase, PERK, followed by eIF2α phosphorylation. Apoptotic module: Activation of caspase 8 and the following BAX/BAK are also required for CRT exposure. Translocation module: CRT is anterogradely transported from the ER lumen to the Golgi apparatus, and then utilize the SNARE dependent exocytosis to present to the cell surface. Recent years, other types of cell death have been shown to induce CRT exposure as well, such as necroptosis and ferroptosis. But the mechanism of how these two types of cell death induce CRT exposure is not well defined yet. We revealed in here that different from the canonical pathway, MeSG doesn’t require the activation of ER stress and caspase 8 to induce CRT exposure. We do show that translocation module is required.
Figure 2. MeSG treatment enhances exposure of calreticulin (CRT) in murine and human melanoma cell line.
(a) B16F10 cells were treated with MeSG at 30 or 100 nM for 3, 12, and 24 hours, or with vehicle control DMSO for 24 hours, or 10,000 rads gamma irradiation. CRT exposure was determined by flow cytometry among viable cells (7AAD negative). Histograms of cell surface CRT MFI (mean fluorescence intensity) are shown. (b) MFI of ecto-CRT from treated cells relative to vehicle shown as Mean ± SD (n = 3). Significance was determined by One-way ANOVA with Tukey correction (*p ≤ .05, ** p ≤ .01, ****P ≤ .0001). (c) Surface CRT on B16F10 cells treated as above were visualized by ImageStream. Fluorescence intensity of ecto-CRT is reported by numbers in yellow. (d,e) UACC903 cells were treated with MeSG at indicated concentrations for 12 or 24 hours, or with vehicle control DMSO for 24 hours. CRT exposure was determined as described before and presented in the histograms (D). MFI of ecto-CRT are shown as Mean ± SD (n = 3) in the line graph. Significance was determined by One-way ANOVA with Tukey correction. (f) B16F10 cells were treated with MeSG (30 nM) for 12 or 24 hours or treated with MeSG for 12 hours at first then cultured in only media for additional 12 hours. CRT exposure was determined. MFI of ecto-CRT are shown as Mean ± SD (n = 3) in the bar graph.
Figure 3. MeSG enhances phagocytosis of B16F10 by murine BMDCs.
(a) Diagram of phagocytosis assay:①Bone marrow cells were isolated from the tibias and femurs of C57BL/6 mice. The bone marrow cells were then treated with GM-CSF for 10 days to be differentiated into bone marrow derived dendritic cells (BMDCs). At the end of incubation, the cells were harvested and stained with the indicated cell surface markers to quantify their differentiation.②B16F10 tumor cells were stained with CellTracker Green for 24 hours, then treated with DMSO,100 nM MeSG or 2.5 µM DOX for 24 hours.③CellTracker Green stained B16F10 tumor cells were co-cultured BMDCs for 5 hours. Then the surface of BMDCs was labeled with APC-conjugated anti-CD11C antibody. Phagocytosis was measured and representative images captured by ImageStream. (b) Representative images of phagocytosis induced by indicated treatments. B16F10 derived material is shown in green (FITC), CD11c staining is shown in red. BF: brightfield. DOX:doxorubicin. (c) The frequency of phagocytic events is quantified (n = 2 replicates).
Figure 4. MeSG treatment doesn’t induce exposure of CD47 and ERp57 on surface of B16F10 and UACC903 cells.
(a,b) B16F10 cells were treated with MeSG at 100 nM, or with vehicle control DMSO for 24 hours. Cell surface CD47 was determined by flow cytometry among viable cells (7AAD negative) and presented in histograms. MFI of surface CD47 were shown as Mean ± SD (n = 3) in bar graph. Significance was determined by One-way ANOVA with Tukey correction.(c,d) B16F10 cells were treated with MeSG at 100 nM, or with DMSO, for 24 hours, or 10,000 rads gamma irradiation. Cell surface ERp57 was determined by flow cytometry among viable cells (7AAD negative) and presented in histograms. MFI of surface ERp57 are shown as Mean ± SD (n = 3) in bar graph. Significance was determined by One-way ANOVA with Tukey correction. (e) UACC903 cells were treated with MeSG at 100 nM, or with DMSO, for 24 hours. Cell surface ERp57 levels were measured by flow cytometry. The quantification of ERp57 MFI are reported as Mean ± SD (n = 3) in bar graph. Significance was determined by t-test.
Figure 5. The effects of MeSG on ER stress related-proteins and PERK phosphorylation.
(a-d) B16F10 cells were treated with MeSG at 100 nM for 3, 6, 9, 12, and 24 hours, or with positive control Thapsigargin (Thap, 5 μM), or with vehicle control DMSO for 24 hours.The levels of CRT, total and phosphorylated PERK, total and phosphorylated eIF2α were determined by Western blotting. Results were quantified by BioRad ImageLab software and presented as Mean ± SD (n = 3) in bar graph. (e-h) UACC903 cells were treated with MeSG 100 nM for 3, 6, 9, 12, and 24 hours, or with positive control Thapsigargin (Thap, 5 μM), or with vehicle control DMSO for 24 hours.The levels of CRT, total and phosphorylated PERK, total and phosphorylated eIF2α were determined by Western blotting. Results were quantified by BioRad ImageLab software and presented as Mean ± SD (n = 3) in bar graph.Statistical significance was determined by One-way ANOVA with Tukey correction (*p ≤ .05, ** p ≤ .01, ****P ≤ .0001).
Figure 6. The relationship of MeSG-induced CRT exposure to ER stress and PERK phosphorylation.
(a,b) B16F10 cells were pre-treated with indicated concentrations of ER stress inhibitor TUDCA (0.5, 1, 1.5 mM) for 2 hours and then co-treated with 30 nM MeSG and TUDCA or vehicle for additional 24 hours. CRT exposure was determined as described before. MFI of surface CRT were presented as Mean ± SD in bar graph. (c,d) B16F10 cells were pre-treated with indicated concentrations of PERK kinase activity inhibitor GSK2606414 (PERKi at 2.5, 5.5, 10 µM) for 2 hours and then co-treated with 30 nMMeSG and PERKi or with vehicle for additional 24 hours. CRT exposure was determined as described before. MFI of surface CRT were presented as Mean ± SD in bar graph. (e-h) B16F10 cells were separately pre-treated with ER stress inhibitor TUDCA (0.5 mM) or PERKi(5.5 μM) for 2 hours then co-treated with MeSG (at 30,100 nM) and TUDCA or MeSG and PERKi or DMSO for 24 hours. The total expression level of CRT, phosphorylation ratio of PERK and eIF2α were determined by Western blotting. Results were quantified by BioRad ImageLab software and presented as Mean ± SD in Bar graph. (i) UACC903 cells were pre-treated with indicated concentrations of PERKi for 2 hours and then co-treated with 6.5 nM MeSG and PERKi or with vehicle for additional 24 hours. CRT exposure was determined as described before. MFI of surface CRT were presented as Mean ± SD in bar graph. (j-m) UACC903 cells were pre-treated with PERKi 5.5 μM for 2 hours then co-treated with 6.5 or 100 nMMeSG and PERKi or DMSO for 24 hours. The total expression level of CRT, phosphorylation ratio of PERK and eIF2α were determined by Western blotting. Results were quantified by BioRad ImageLab software and presented in Bar graph as Mean ± SD.Statistical significance were all determined by One-way ANOVA with Tukey correction (*p ≤ .05, ** p ≤ .01, ****P ≤ .0001).
Figure 7. PERK and ERp57 knockdown to explore their role in MeSG induced CRT exposure.
(a-c) B16F10 cells were transfected with PERK siRNA (siPERK) or scramble RNA (scRNA) for 48 hours, then treated with 100 nM MeSG, 5 µM thapsigargin or DMSO for 24 hours. The expression levels of total and phosphorylated PERK, total and phosphorylated eIF2α were determined by Western blotting. Results were quantified by BioRad ImageLab software and presented as Mean ± SD (n = 3) in bar graph (d) Ecto-CRT of cells treated as described in (a) were determined by flow-cytometry. MFI of surface CRT were presented as Mean ± SD in bar graph. (e)Cell surface CRT images were captured by ImageStream. Geometric mean of surface CRT is reported by numbers in yellow. (f-h) UACC903 cells were transfected with PERK siRNA (siPERK), ERp57 (siERp57) or scramble RNA (scRNA) for 24 hours, then treated with 100 nM MeSG or DMSO for 24 hours. The expression levels of indicated protein were determined by Western blotting. Quantification of Western blotting results is presented as Mean ± SD in the bar graph. (l) Cell surface CRT were measured by Flow cytometry. MFI of surface CRT were presented as Mean ± SD in bar graph.All statistical significance were determined by One-way ANOVA with Tukey correction (*p ≤ .05).
Figure 8. MeSG induce apoptosis in UACC903 cells before CRT exposure.
(a) UACC903 cells were treated with MeSG at 1,10,100 nM for 12, 24, 36 and 48 hours, then stained with Annexin-V and 7-AAD to measure apoptosis level. Quantification of percentage of cells at 4 stages during apoptosis was presented as Mean ± SD in bar graph. Statistical significant were determined by TWO-WAY ANOVA with Tukey’s multiple comparisons test. (b) Ecto-CRT of UACC903 treated in A were determined by flow cytometry. MFI of ecto-CRT from treated cells relative to vehicle were shown as Mean ± SD in bar graph. Statistical significance was determined by One-way ANOVA with Tukey correction. (c,d) B16F10 cells were treated with MeSG 100 nM for 12, 24, 36 and 48 hours or with DMSO, then stained with Annexin-V and 7-AAD to measure apoptosis level. Distribution of cell population in apoptosis procedure was presented in dot plot, and the quantification is shown as Mean ± SD in bar graph.Statistical significant were determined by TWO-WAY ANOVA with Tukey’s multiple comparisons test.
Figure 9. Inhibition of caspase activation can rescue cells from apoptosis induced by MeSG but can not inhibit CRT exposure.
(a) UACC903 cells were treated with MeSG at 6.5 nM or 100 nM or co-treated with pan-caspase inhibitor Z-VAD-FMK at 25uM or caspase-8 specific inhibitor Z-IETD-FMK at 20uM or for 24 hours, then stained with Annexin-V and 7-AAD to measure apoptosis level. Distribution of cell population in apoptosis procedure is shown as Mean ± SD in bar graph. Statistical significant was determined by TWO-way ANOVA with Tukey’s multiple comparisons test. (b) Ecto-CRT of UACC903 treated in E were measured and MFI of ecto-CRT from treated cells relative to vehicle were shown as Mean ± SD in bar graph. Statistical significance was determined by One-way ANOVA with Tukey correction (c,d) B16F10 cells were treated with MeSG at 100 nM or co-treated with Z-IETD-FMK or Z-VAD-FMK for 24 or 48 hours, then stained with Annexin-V and 7-AAD to measure apoptosis level. Distribution of cell population in apoptosis procedure was presented in dot plot C, and the quantification was shown as Mean ± SD in bar graph D. Statistical significant was determined by TWO-way ANOVA with Tukey’s multiple comparisons test. (e) Ecto-CRT of B16F10 cells treated in G were measured as mentioned before. MFI of ecto-CRT from treated cells relative to vehicle shown as Mean ± SD (n = 3) in bar graph.Statistical significance was determined by One-way ANOVA with Tukey correction (*p ≤ .05, ** p ≤ .01, ****P ≤ .0001).
Figure 10. Induction of calreticulin exposure by MeSG can be blocked by Brefeldin A pre-treatment.
(a,b) UACC903 cells were pre-treated with BFA at 2, 5 and 10 μM for 2 hours, then treated with 100 nM MeSG for 24 hours. Ecto-CRT were measured as mentioned before and presented in the histograms, quantification was presented as Mean ± SD in bar graph (n = 3). (c) B16F10 cells were pre-treated as UACC903 cells in A,B. Ecto-CRT were quantified and shown as Mean ± SD in bar graph (n = 3).Statistical significance was determined by One-way ANOVA with Tukey correction, ****P ≤ .0001.
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