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. 2021 Dec 24;11:24414. doi: 10.1038/s41598-021-03618-9

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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PP2ACA-knock-down H-17 cells are hypersensitive to bacterial lipoproteins and are useful for detecting mycoplasma contamination. (A) Western blots showing PP2ACA depletion in H-17 PP2ACA^KD clones 25 and 28 compare to H-17 cells. See also Supplementary Fig. S5A for uncropped, full-size original Western Blot. (B) Representative flow cytometry histograms for TLR2 from H-17, H-17 PP2ACA^KD_25 and H-17 PP2ACA^KD_28 and percentage of positive cells are indicated on corresponding panels. Histograms are normalised to the mode. (C) Quantification of mCFP expression in H-17 PP2ACA knock-down compared to scrambled upon stimulation with Pam3CSK4 (100 ng/ml) for 6 h. Bar graphs represent MFI ± SEM (n = 4). (D) Western blots showing increasing expression of mCFP in H-17 PP2ACA^KD_25, H-17 PP2ACA^KD_25 compared to H-17 Scrambled after treatment with Pam3CSK4 (100 ng/ml) for 2 h. See also Supplementary Fig. S5B for uncropped, full-size original Western Blot. (E) Representative flow cytometry histograms of mCFP expression in H-17 PP2ACA^KD_25 [grey, solid line] compared to H-17 SOCS1^−/− [green, dashed line] after treatment for 2 h or 3 h with Pam3CSK4 (100 ng/ml). Histograms are normalised to the mode. Unfilled histograms represent unstimulated cells. (F) Quantification of mCFP expression in H-17 PP2ACA^KD compared to H-17 SOCS1^−/− after treatment for 1 h, 2 h or 3 h with Pam3CSK4 (100 ng/ml). Bar graphs represent MFI ± SEM (n = 4). (G) PCR analysis of a mycoplasma-positive (Sample #1) and a mycoplasma-negative (Sample #2) cell culture supernatant. See also Supplementary Fig. S5C for uncropped, full-size original agarose gel. (H) Representative flow cytometry histograms of mCFP expression in H-17 (left panels) or H-17 PP2ACA^KD_25 cells (right panels) after 3 h of exposure to cell culture supernatants from (G). Incubation for 3 h with Pam3CSK4 (100 ng/ml) served as positive control. Histograms are normalised to the mode and the number indicates the mean fluorescent intensity (MFI).