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1 Roles of endoplsmic reticulum stress-medited poptosis in -polrized mcrophges during mycocteril infections Supplementry informtion Yun-Ji Lim, Min-Hee Yi, Ji-Ae Choi, Jung-hwn Lee, Ji-Ye Hn, Sung-Hee Jo, Sung-Mn Oh, Hyun Jin Cho, Dong Woon Kim, Min- Woong Kng nd Chng-Hw Song Supplementry Figure GM-CSF M-CSF LPS IFN-γ TNF-α IL IL Notch inos TNFα IL-p4 Arginse CD6 Fizz chi3l3 β-ctin NO /NO 3 production (um) 6 4 ** M Reltive intensity Fizz Arginse TNFα Notch GM-CSF M-CSF inos IL-p4 CD6 chi3l c IL-p4 (ng/ml)..5 IL-6 (ng/ml) LPS IFN-γ TNF-α IL IL LPS IFN-γ TNF-α IL IL M M TNF (ng/ml).. MCP- (ng/ml).. IL- (ng/ml).4. M M M
2 Supplementry figure, relted to Figure. Cytokine-induced mcrophge polriztion () BMDMs were treted with LPS, IFN-γ, nd TNF-α ( stimulus) or IL-4 nd IL-3 ( stimulus). Twenty-four hours lter, mcrophge mrna ws hrvested nd susequently nlysed for indicted mcrophge polriztion mrker genes y qpcr (Left). Reltive intensities of the nds nlysed y quntittive densitometry re shown (Right). ( nd c) Using superntnt frctions from cell cultures, () NO production, nd (c) IL-p4, IL-6, TNF, MCP-, nd IL- cytokine secretions were mesured. Dt re representtive of three independent experiments (n=3). Sttisticlly significnt differences re indicted; *p <.5, ** p <. nd p <.. M=unstimulted mcrophges.
3 Supplementry Figure h h p-stat STAT p-stat3 STAT3 NO /NO 3 production (um) 5 5 Supplementry figure, relted to Figure. Incresed -phenotype mrkers in H37R-infected Rw64.7 cells () Rw64.7 cells were polrized into nd phenotypes, s descried in Figure S, nd then or mcrophges were infected with H37R for h. Cell lystes were nlysed for STAT, p-stat, STAT3, nd p- STAT3 using western lot. () Using superntnt frctions from cell cultures, NO production ws mesured. Dt re representtive of three independent experiments (n=3). Sttisticlly significnt differences re indicted; *p <.5, ** p <. nd p <..
4 Supplementry Figure 3 WT TLRKO TLR4KO p-stat TLR TLR4 β-ctin STAT p-stat3 STAT3 c 3 3 d IL-p4 (ng/ml) IL- (ng/ml) TLR MyD88 β-ctin WT TLR4KO WT TLR4KO Supplementry figure 3, relted to Figure. TLR4 Signling is not involved in modultion of mcrophge polriztion during Mt H37R infection () Genotyping of TLR- or TLR4-deficient mice ws performed using qpcr with specific primers. (-c) BMDMs from WT, TLR-, nd TLR4-deficient mice were infected with H37R for indicted time. These cells were nlysed for () p-stat nd p-stat3 using western lot. Using superntnt frctions from cell cultures, (c) IL-p4 nd IL- cytokine secretions were mesured. (d) Genotyping of TLR- or MyD88-deficient mice ws performed using qpcr with specific primers. These results re representtive of t lest three experiments.
5 Supplementry Figure 4 CFUx 5.5 Con. PEITC * CFUx Con. NB CFUx 5.5 Con. CE ** CFUx sicon. siklf4 ** Supplementry figure 4, relted to Figure 5. Mcrophge polriztion controls intrcellulr survivl of H37Rv () nd mcrophges were pre-treted with inducers for h s follows: mcrophges received NICD inducer (PEITC,.5 μm) nd mcrophges received KLF4 inducer (NB, mm). nd cells were then infected with H37Rv for or h. Intrcellulr survivl of H37Rv ws mesured y CFU enumertion. () mcrophges were pre-treted with γ-secretse inhiitor (compound E, 5 μm) for h. mcrophges were trnsfected with KLF4 sirna ( nm) for h. Then, nd cells were infected with H37Rv for or h nd nlysed for the intrcellulr survivl of Mt. Sttisticlly significnt differences re indicted; *p <.5, ** p <. nd p <.. M=unstimulted mcrophges.
6 Supplementry Figure 5 PBS p-stat Dy (LPS.5 μg + IFNγ.5 μg in 4μl PBS) (IL-4.5 μg + IL-3.5 μg in 4μl PBS) (IFNγ.5 μg in 4μl PBS) (IL-4.5 μg in 4μl PBS) Scrifice R infection STAT p-stat3 STAT3 Supplementry figure 5, relted to Figure 6. Estlishment of n in vivo mouse model to study mcrophge polriztion () C57BL/6 mice were intrnslly dministered LPS nd IFN-γ, or IL-4 nd IL-3 s follows: For -like phenotype,.5 μg of LPS nd.5 μg IFNγ in 4 μl PBS were dministered; nd for -lilke phenotype,.5 μg of IL-4 nd.5 μg IL-3 in 4 μl PBS were dministered ccording to the protocol, which ws descried in detil in method section. () After dministering the stimulnts, the mice were infected with H37R (5x 6 ) for 3 dys. Whole-cell proteins were extrcted y homogenizing the lungs in lysis uffer. STAT nd STAT3 phosphoryltion ws determined y western lot. Totl STATs were used for cell loding controls. These results re representtive of t lest three independent experiments.
7 Supplementry Figure 6 Supplementry figure 6, Schemtic representtion of mcrophge polriztion during mycocteril infection This illustrtion reflects tht phenotypic nd functionl chrcteristics of mcrophges re different in (upper) nd (lower) mcrophges during Mt infection. mcrophges prove eneficil for removing intrcellulr survivl of Mt vi ER stress-induced poptotic pthwy, in contrst, mcrophges pper s fvorle niche for long-term persistence of Mt.
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