(A) Smurf1 knockdown in primary human monocyte-derived macrophages transduced with controls or two different SMURF1 shRNA constructs. mRNA levels of SMURF1 were analyzed by quantitative RT-PCR. Bars represent mean ± SEM (normalized to control scrambled shRNA). Similar results were observed in three independent experiments. ****P<0.0001; t-test.
(B) Mtb growth in primary human monocyte-derived macrophages transduced with control scrambled or SMURF1shRNA. Infected cells were lysed at the indicated time-points and Mtb growth was determined by counting CFUs. Bars are mean ± SEM of quadruplicate samples; each sample was normalized to day 0. Similar results were observed in two independent experiments. See Figure S5A for results from one additional donor. *P<0.05; **P<0.01 for indicated comparison; two-way ANOVA.
(C) Immunohistochemical staining for SMURF1 in lung biopsies of two human patients with active pulmonary tuberculosis or from a normal lung. Scale bars, 100 μm.
(D) Immunofluorescence staining of a lung biopsy of a human patient with active pulmonary tuberculosis using anti-Mtb and anti-SMURF1 antibodies. Left panel, image of Mtb that colocalizes with SMURF1. See Figure S5 for additional examples. Right panel, image of Mtb that do not colocalize with SMURF1. Scale bar, 5 μm. See also Figure S5.