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. 2021 Jul 15;89(8):e00141-21. doi: 10.1128/IAI.00141-21

FIG 4.

FIG 4

Expression of TLR2, NOD1, and EGFR in B. henselae-infected MSCs. (A) TLR2, TLR4, NOD1, and EGFR mRNA expression levels in uninfected (CTRL) and B. henselae-infected MSCs were determined by quantitative PCR and normalized to that of RPL13A. Data are expressed as mean ± SEM of four independent experiments. *, P < 0.05, unpaired t test. (B) TLR2 and TLR4 protein expression levels on MSC membranes were analyzed by FACS in MSCs at 4 days pi. Cells were immunostained with anti-TLR2, anti-TLR4, or specific isotype control antibodies. The percentages of positive cells are indicated in each quadrant. Fluorescence minus one (FMO) controls for the antibodies are shown as inset in the corresponding dot plot. Data are representative of three independent experiments (left and middle) or as mean ± SEM (right). (C) Cell extracts from MSCs infected with B. henselae for 30, 60, or 120 min or with human EGF (50 ng/ml) for 15 min were subjected to immunoblotting using anti-phospho-Y1068-EGFR or anti-EGFR antibodies. (D) Analysis of CXCL8 in the supernatants from uninfected or B. henselae-infected MSCs that were pretreated or not for 6 h with a neutralizing anti-TLR2 antibody (10 μg/ml) (n = 6 experiments) (upper) or with the EGFR inhibitor gefitinib (10 μM) or the RIPK2 inhibitor GSK583 (GSK) (1 μM) (n = 4 experiments) (lower) and then stimulated for 96 h. Data are shown as the percentage (mean ± SEM) of CXCL8 production, compared to the specific isotype control antibody or DMSO set as 100%. *, P < 0.05 versus B. henselae-infected cells, unpaired t test. (E) To evaluate B. henselae internalization, MSCs were pretreated for 6 h with the neutralizing anti-EGFR antibody (10 μg/ml) (n = 3 independent experiments) (upper) or gefitinib (10 μM) (n = 4 independent experiments) (lower), and CFU values for intracellular bacteria, determined after 1 and 2 days of incubation, are expressed as percentages, relative to the CFU of the specific isotype control antibody- or DMSO-treated cells set as 100%. Data are shown as mean ± SEM. *, P < 0.05 versus internalized bacteria in untreated cells, unpaired t test.