www.pnas.org/cgi/doi/10.1073/pnas.1813458115 2 Lee et al. Supplementation i nformati on Supplemental Materials and Methods Materials. DMEM, RPMI 1640, penicillin/streptomycin, and trypsin-EDTA were purchased from Invitrogen. Sheep EA’s targets and GVB ++ buffer were purchased from Berlex. Versene was obtained from Lonza. LPS was purchased from Sigma (Escherichia coli 0127:B8). Rapamycin was purchased from Sigma. The Adenovirus expression vector for mature SREBP-1a was described previously (1 ). Animals. The original SREBP-1a-deficinet mouse line (SREBP-1aDF) was described previously ( 2 ) but as reported by Gerlic et al. (3) and Im et al. (4), the SREBP-1a gene trap was inserted into the Srebf1 locus on chromosome 11 within the genome of B129 strain. The Nlrp1 locus is tightly linked to the Srebf1 locus and after 10 generations of backcrossing to C57Bl6J, the mice still had the Nlrp1 locus from B129. Because the Nlrp1a gene from the B129 locus is not expressed for an unknown molecular reason these mice did not express Nlrp1a. Knowing this limitation, we subjected the mice to several more rounds of backcrossing to facilitate recombination to generate the SREBP-1aDF/B6 mice used in the current study, which contains the Nlrp1 locus from C57Bl6J mice. Mice with floxed alleles for raptor on C57BL6J background (5) and SCAP on a mixed B6/129 (6) were obtained from JAX. The SCAP floxed mice were backcrossed with C57BL6/J mice for 12 generations. The myeloid specific knockouts for both raptor and SCAP were generated by mating into the C57BL6J strain of LysM6 CRE mice. Knockout and control littermate floxed mice were used in all experiments. Mice were maintained in 12 h light/12 h dark cycles with free access to food and water in pathogen free mouse facility. Mice were harvested for bone marrow isolation at the end of the dark cycle. All procedures were performed in accordance with the Institutional of Animal care and Use Committees at the Sanford Burnham Prebys Medical Discovery Institute and the Keimyung University School of Medicine, Daegu, South Korea (KM-2015-32R3). Phagocytosis assay. Phagocytosis assays were performed using slight modifications to a previously published procedure (8). Opsonized target particles for the phagocytosis assay were sheep erythrocytes (E) with either IgG anti-sheep red blood cells (EA IgG ) prepared as
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www.pnas.org/cgi/doi/10.1073/pnas.1813458115
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Lee et al. Supplementation information Supplemental Materials and Methods
Materials. DMEM, RPMI 1640, penicillin/streptomycin, and trypsin-EDTA were purchased
from Invitrogen. Sheep EA’s targets and GVB++ buffer were purchased from Berlex.
Versene was obtained from Lonza. LPS was purchased from Sigma (Escherichia coli
0127:B8). Rapamycin was purchased from Sigma. The Adenovirus expression vector for
mature SREBP-1a was described previously (1 ) .
Animals. The original SREBP-1a-deficinet mouse line (SREBP-1aDF) was described
previously ( 2 ) but as reported by Gerlic et al. (3) and Im et al. (4), the SREBP-1a gene
trap was inserted into the Srebf1 locus on chromosome 11 within the genome of B129 strain.
The Nlrp1 locus is tightly linked to the Srebf1 locus and after 10 generations of backcrossing
to C57Bl6J, the mice still had the Nlrp1 locus from B129. Because the Nlrp1a gene from
the B129 locus is not expressed for an unknown molecular reason these mice did not
express Nlrp1a. Knowing this limitation, we subjected the mice to several more rounds of
backcrossing to facilitate recombination to generate the SREBP-1aDF/B6 mice used in the
current study, which contains the Nlrp1 locus from C57Bl6J mice. Mice with floxed alleles
for raptor on C57BL6J background (5) and SCAP on a mixed B6/129 (6) were obtained from
JAX. The SCAP floxed mice were backcrossed with C57BL6/J mice for 12 generations. The
myeloid specific knockouts for both raptor and SCAP were generated by mating into the
C57BL6J strain of LysM6 CRE mice. Knockout and control littermate floxed mice were used
in all experiments. Mice were maintained in 12 h light/12 h dark cycles with free access to food
and water in pathogen free mouse facility. Mice were harvested for bone marrow isolation
at the end of the dark cycle. All procedures were performed in accordance with the
Institutional of Animal care and Use Committees at the Sanford Burnham Prebys Medical
Discovery Institute and the Keimyung University School of Medicine, Daegu, South Korea
(KM-2015-32R3).
Phagocytosis assay. Phagocytosis assays were performed using slight modifications to a
previously published procedure (8). Opsonized target particles for the phagocytosis assay
were sheep erythrocytes (E) with either IgG anti-sheep red blood cells (EAIgG) prepared as
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previously described (7). Briefly, eight-well Lab Tek chambers (Nalge Nunc Intl., Naperville, IL)
were coated with human serum albumin (HSA) (MDI/American Red Cross, Louisville, KY) as
a control protein. Phagocytic cells resuspended in phagocytosis buffer (RPMI, supplemented
with 2 mM L-glutamine, 10 µg/ml pen/strep, 5 mM MgCl2) are added to each chamber, the
cells centrifuged at 700 rpm (RT6000, Dupont Sorvall) for 3 min and subsequently placed at
37 °C in 5% CO2 for 30 min. Targets were then added (106/100 µl), the slides again
subjected to centrifugation (700 rpm, 3 min), and incubated for 30 min at 37 °C. After
removing unbound targets by washing, bound, uningested targets were removed by
hypotonic lysis ( 8 ) (). Cells were then fixed in freshly diluted 1% glutaraldehyde and stained
with GIMSA (Sigma). Phagocytosis was quantitated using light microscopy. The number of
E-targets ingested per 200 effector cells was defined as the phagocytic index (PI), whereas
the percentage of effector cells ingesting at least one E-target was defined as the percent
phagocytosis. Over 200 effector cells were scored per well and duplicate sample wells per
condition were used for each experiment.
Isolation of BMDMs. Bone marrow-derived macrophages were isolated from C57B/6J,
SREBP-1aDF/B6, SCAPfl/fl, SCAP LysM6-SCAP KO (SCAP mKO), Raptorfl/fl, or LysM6-
Raptor KO (raptor mKO) mice. Femurs and tibias were isolated and flushed with DMEM
supplemented with 2% FBS. After RBC were lysed using ACK lysis buffer (0.15 M NH4Cl, 10
mM KHCO3, and 0.1 mM Na2-EDTA, pH 7.2), macrophages were depleted by a pre-
adhesion step for 2 h at 37°C in 5% CO2. Following the differentiation for 7 days in culture,
BMDMs were subcultured to use in experiments.
Adenovirus infection in BMDMs. Macrophages were plated in 100 mm dishes (1 x
106 cells/dish). Ad-SREBP-1a (10 multiplicities of infection [moi]) was added to cells in DMEM medium containing 20% L929 conditioned medium without FBS at 37°C with 5% CO2 for 6
h., and medium with FBS was added. After incubating for additional 24 h, cells were collected
for analysis.
Isolation of RNA and qPCR. Total RNA was prepared from macrophages of WT and
SREBP-1aDF/B6 mice by the Trizol procedure (Invitrogen) and cDNA was synthesized
using cDNA superscript kit (Bio-Rad) to use for qPCR with CFX96 Bio-Rad qPCR machine
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(Bio-Rad). mRNA levels were normalized for expression of ribosomal protein L32 mRNA as
control and calculated by the comparative threshold cycle method.
Immunoblotting. Proteins were isolated from BMDM and immunoblotting was performed
following a modified protocol of previous method (9). Proteins were resolved by 5-10% Tris-
HCl SDS/PAGE gel electrophoresis and transferred onto nitrocellulose (Bio-Rad). Antibodies
to the following proteins were purchased from Cell Signaling Technology (Danvers, MA,