Calreticulin surface exposure is abrogated in cells lacking, chaperone-mediated
autophagy-essential gene, LAMP2A
Abhishek D. Garg, Aleksandra M. Dudek and Patrizia Agostinis
Supplementary Figure S1 (Legend):
Wild type 3T3 fibroblast cells (i.e. LAMP2A+/+ cells) and 3T3 fibroblast cells lacking the chaperonemediated autophagy (CMA)-essential gene, LAMP2A (i.e. LAMP2A-/- cells) were cultured at 37°C under
5% CO2 in DMEM (containing 0.11 g/L sodium pyruvate and 4.5 g/L glucose) supplemented with 2 mM
glutamine, 100 units/ml penicillin, 100 µg/L streptomycin and 10% fetal bovine serum. As applicable, the
respective 3T3 cells were in one case treated with mitoxantrone (MTX from Sigma; 1 µM) and recovered
4 h post-treatment for all the assays. In the other case for hypericin-based photodynamic therapy (HypPDT), the respective 3T3 cells were pre-incubated with 200 nM hypericin (for 2 h in serum-free culture
medium) followed by light irradiation-based “excitation” of hypericin (fluence - 1.35 J/cm2). Hyp-PDT
treated cells were recovered 15 min post-treatment for all the assays. Hypericin was prepared and light
irradiation was performed as described previously.1, 2 The respective post-treatment recovery time points
used for experimentation were confirmed to be associated with non-significant plasma membrane
permeabilization via LDH release assay as described previously.1, 2
(A) Absence of LAMP2A gene does not affect Hyp-PDT or MTX-induced ATP secretion.
LAMP2A+/+ and LAMP2A-/- cells were left untreated (CNTR) or treated with Hyp-PDT or MTX and
recovered post-treatment, as described above. The resulting (serum-free) conditioned media was analyzed
for the presence of extracellular ATP. Extracellular ATP was measured via an ATP assay mix (Sigma)
based on luciferin-luciferase conversion, following the manufacturer’s instructions. Bioluminescence was
assessed by optical top reading via a FlexStation 3 microplate reader (Molecular Devices Inc., California,
USA). Data are presented as relative light unit (RLU) values (3 independent experimental determinations;
mean ± s.e.m.; Student’s t-test was used for statistical analysis - *P<0.05 vs. respective CNTRs, N.S. =
not significant, as indicated by the bars).
(B) Absence of LAMP2A gene abrogates basal, Hyp-PDT or MTX-induced Calreticulin (CRT)
surface exposure. LAMP2A+/+ and LAMP2A-/- cells were left untreated (CNTR) or treated with HypPDT or MTX and recovered post-treatment, as described above. Following this, the cell surface proteins
were biotinylated as described previously.1, 2 For CNTR samples, one set of samples were exposed to the
buffer containing biotin (denoted by ‘+BIO’) while the other set was exposed to only the buffer as a
negative control (denoted by ‘-BIO’). The capture of plasma membrane proteins was confirmed by
detecting CD91 in the biotinylated protein fractions. In all the figures (as applicable), ‘Plasma membrane
proteins’ are proteins in the biotinylated surface fraction while ‘intracellular proteins’ are the unbound
proteins that are non-biotinylated. This was followed by immunoblotting as described previously3;
utilizing antibodies against CRT (Stressgen, Canada), actin (Sigma) and antibody (1704) recognizing
CD91 protein4. Secondary antibodies conjugated to horseradish peroxidase were purchased from Cell
Signaling Technology (USA).
(C) Knocking-down ATG5 in cells lacking LAMP2A gene does not restore basal, Hyp-PDT or
MTX-induced CRT surface exposure. LAMP2A+/+ and LAMP2A-/- cells were both either transfected
with scrambled (Scr)-siRNA or an ATG5-specific siRNA respectively, by adding 500 µL serum-free
1
culture media with 10 µL Dharmafect (Thermo Fisher Scientific, Belgium) and 5 µL ATG5 or Scr siRNA
(20 µM, Dharmacon, Thermo Fisher Scientific) to culture dishes with 2 mL serum-free culture media
(final concentration of 40 nM siRNA). Here, 3 h following transfection, 2.5 mL culture media containing
5% FBS was added and the experiments were carried out 48 h after transfection. Knock-down of ATG5
in the respective conditions was confirmed via immunoblotting utilizing antibodies against ATG5
(Abcam, UK) and Actin (Sigma) (Left). Subsequently, the respective Scr or ATG5-siRNA transfected
LAMP2A+/+ or LAMP2A-/- cells respectively, were left untreated (CNTR) or treated with Hyp-PDT or
MTX and recovered post-treatment, as described above (Right). Following this, the cell surface proteins
were biotinylated as described in (B). The denotations and immunoblotting procedures are the same as
described for (B).
References:
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2.
Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T, et al. A novel pathway combining
calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J 2012, 31(5): 10621079.
3.
Vantieghem A, Assefa Z, Vandenabeele P, Declercq W, Courtois S, Vandenheede JR, et al. Hypericininduced photosensitization of HeLa cells leads to apoptosis or necrosis. Involvement of cytochrome c and
procaspase-3 activation in the mechanism of apoptosis. FEBS Lett 1998, 440(1-2): 19-24.
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Reekmans SM, Pflanzner T, Gordts PL, Isbert S, Zimmermann P, Annaert W, et al. Inactivation of the
proximal NPXY motif impairs early steps in LRP1 biosynthesis. Cell Mol Life Sci 2010, 67(1): 135-145.
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