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cmi12133-sup-0001.tif716K

Fig. S1.SULF1 and SULF2 expression in Hela cells. Total RNA was isolated from HeLa cells. SULF1 and SULF2 mRNA levels were quantified by qRT-PCR analysis and normalized to GAPDH mRNA.

cmi12133-sup-0002.tif13482K

Fig. S2. Overexpression of SULF1 or SULF2 in HeLa cells decreases Chlamydia vacuole formation. HeLa cells were transiently transfected with the vector expressing Myc-tag alone (Vector) or Myc-tagged SULF1 (HS1), Myc-tagged SULF2 (HS2), or a catalytically inactive version of Myc-tagged SULF1 (IHS1) and then infected with C. muridarum. At 24 hpi, cells were fixed and stained with antibodies to Myc (red), to visualize intracellular and extracellular SULFs, and to the Chlamydial Major Outer Membrane Protein (MOMP; Green) to visualize vacuoles. Bacterial and host DNA were detected by DAPI staining (blue). White arrows point to cells expressing Myc-tagged HS1, HS2, or IHS1. Note the presence of multiple vacuoles in the vector or IHS1-transfected cells (yellow arrows) and the decreased number of vacuoles per cell in SULF1- and SULF2-transfected cells compared with vector-transfected and IHS-transfected cells (MOMP staining; also see Fig. 1C for quantification). Scale bar = 20 μm.

cmi12133-sup-0003.tif676K

Fig. S3.SULF1 mRNA expression is increased upon SULF2 depletion.

A. Total RNA was isolated from HaCaT cells. SULF1 and SULF2 mRNA levels were quantified by qRT-PCR and normalized to GAPDH mRNA levels.

B. HaCaT cells were transduced with lentivirus encoding scrambled shRNA or two different shRNAs targeting SULF2 (SULF2A and SULF2B) and selected for puromycin resistance. Total RNA was isolated from shRNA-transduced cells and hsulf1 mRNA level was quantified by qRT-PCR. mRNA expression was normalized to GAPDH and then expressed as a fold change compared with scrambled shRNA-transduced cells.

cmi12133-sup-0004.tif4241K

Fig. S4.Chlamydia lung infection leads to inflammatory changes and production of infectious progeny. Wild type mice were intranasally infected with 5 × 106 IFU of C. muridarum.

A. Infectious progeny were recovered from whole lungs at 5 and 48 hpi, stored frozen, and titred by quantification of vacuole formation upon secondary infection of HeLa cells for 24 h. Whereas very few infectious progeny were recovered at 5 hpi, abundant viable progeny were detectable at 48 hpi.

B. H&E staining of representative lung section reveals abundant inflammatory infiltrates in Chlamydia-infected lungs at 48 hpi.

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