EMBO/soda-nel huggingface dataset:
p38 MAPK-dependent phosphorylation of TFEB promotes monocyte to macrophage differentiation (2022)
Figure 3

Figure 3. LPS stimulation induces p38 MAPK-dependent phosphorylation of TFEB-S401 in macrophages.

Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with 1μg/ml LPS for the indicated times.

Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with either 20 μM SB203580 or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS for 30 min.

Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05 and (**)p<0.01 from three independent experiments.

Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg/ml LPS for the indicated times.

Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle (DMSO) or 20 μM SB203580 for 1 h prior to the addition of 1μg/ml LPS for the indicated times.

Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS for 1 h. Immunoblots are representative of at least three independent experiments.

Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments.

Data information: n = 3 biological replicates (each dot represents a biological replicate).
p38 MAPK-dependent phosphorylation of TFEB promotes monocyte to macrophage differentiation (2022)
Figure 4

Figure 4. Activation of TFEB depends on S401 phosphorylation during macrophage differentiation.

Flow chart indicating the different steps followed to differentiate Naïve into macrophage-like THP1 cells.

Immunoblot analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA for the indicated times, PMA-differentiated THP1 (Rested) cells treated without or with 250 nM Torin-1 for 1 h.

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA for the indicated times and PMA-differentiated THP1 (Rested) cells.

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for the indicated times.

Immunofluorescence confocal microscopy of naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 1 h. Scale bars, 10 μm.

Immunoblot analysis of proteins from nuclear fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM Torin-1 for 1 h. Immunoblots are representative of at least three independent experiments.
p38 MAPK-dependent phosphorylation of TFEB promotes monocyte to macrophage differentiation (2022)
Figure 5

Figure 5. Immune genes expression is affected in TFEB-S401A mutant cells.

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 6 h. This condition was used to perform RNA-Seq analysis.

Volcano plot indicating distribution of genes up- (red, shrunkenLog2FC ≥ 1) and down- (green, shrunkenLog2FC ≤ −1) regulated in naïve THP1-WT cells (Control) versus naïve THP1-WT cells incubated with 50 ng/ml PMA for 6 h. Black symbols represent genes with a shrunkenLog2FC between 1 and −1.

MSigDB Hallmark 2020 bar chart showing the top ten enriched terms from RNA-Seq analysis of naïve THP1-WT cells (Control) versus naïve THP1-WT cells incubated with 50 ng/ml PMA for 6 h. The corresponding significant p-values (<0.05) are included with an asterisk (*) indicating that the terms also have significant q-value (<0.05).

Heat map of 50 differentially expressed immune genes from RNA-Seq analysis of naïve THP1-WT cells (WT-PMA) versus naïve TFEB-S401A knock-in (I11-PMA) cells incubated with 50 ng/ml PMA for 6 h.

Relative quantitative RT-PCR analysis of the mRNA expression of chemokines (CXCL1, CXCL3, CXCL8 and CCL5), cytokines (IL1β and LIF), and key immune regulators (IFNGR2 and EREG) genes in naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells incubated with 50 ng/ml PMA for the indicated times. Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 as compared to the same treatment condition in THP1-WT cells from three independent experiments.

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).
p38 MAPK-dependent phosphorylation of TFEB promotes monocyte to macrophage differentiation (2022)
Figure 6

Figure 6. Defect in activation of TFEB-S401A mutant macrophages upon LPS treatment.

Relative quantitative RT-PCR analysis of the mRNA expression of cytokine- (IL1β, IL10, IL18, IL33, IL23A, IL1R and TNFα) and chemokine (CXCL3, CXCL5 and CXCL6) -related genes in PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) incubated with 1 μg/ml LPS for the indicated times. Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 as compared to the same treatment condition in THP1-WT cells from three independent experiments.

Immunoblot analysis of protein lysates and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min.

C-F. Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments.

Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm.

Quantification of the percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments.

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).

Expanded View Figure Legends
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf1

(a,b) Bacteria in the spleens of Slamf1−/−Rag1−/− and Rag1−/− BALB/c mice (a) or Slamf1−/− and Slamf1+/+ BALB/c mice (b) 48 h after intraperitoneal injection of virulent S. typhimurium 14028s or attenuated S. typhimurium SseB−. CFU, colony-forming units; ND, not detectable. Data are representative of four independent experiments (mean and s.d.). (c–e) Killing of bacteria by peritoneal macrophages from Slamf1+/+ and Slamf1−/− BALB/c mice exposed to S. typhimurium SseB− (c), E. coli F18 (d) or S. aureus (e), assessed by gentamycin assay. Data are representative of five independent experiments (mean and s.d.). (f) Uptake of bacteria by Slamf1+/+ (red solid lines) or Slamf1−/− (black solid lines) BALB/c peritoneal macrophages incubated at 37 °C with E. coli–eGFP or S. typhimurium–eGFP or by Slamf1+/+ macrophages incubated for 60 min at 4 °C with the bacteria (dotted lines). Right, mean fluorescence intensity (MFI). Data are representative of three independent experiments (mean and s.d.).
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf2

(a) NOX2 activity in Slamf1+/+ and Slamf1−/− BALB/c peritoneal macrophages stimulated for 0–100 min with E. coli F18, S. aureus or PMA, assessed with lucigenin. Data are representative of five independent experiments (mean ± s.d.). (b) Phagosomal pH of Slamf1+/+, Slamf1−/− and gp91phox−/− B6 primary macrophages loaded for 0–200 min with pHrodo-coated E. coli or S. aureus, analyzed by flow cytometry. Data are representative of three independent experiments (mean ± s.d.). (c) NOX2 activity in primary macrophages in response to LPS, purified OmpC, peptidoglycan (PGN) or PMA, assessed with lucigenin. TLR4-KO, TLR4-deficient (strain del/Jtht; C3H). Data are representative of five independent experiments (mean ± s.d.).
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf3

(a) Fluorescence microscopy of E. coli–containing phagosomes in primary macrophages transfected with RFP-conjugated LAMP-1, showing colocalization with E. coli–eGFP or S. aureus–eGFP. Right, quantification of LAMP-1+ phagosomes in the microscopy at left. (b) Immunoblot analysis of LAMP-1 in phagosomes isolated by sucrose-gradient flotation from RAW264.7 macrophages after phagocytosis by Slamf1- or mock-transfected RAW264.7 macrophages of beads coated with E. coli outer membrane extract. WCL, whole-cell lysate. Right, quantification of LAMP-1 in the immunoblot at left. (c) Localization of MHC class II–eGFP (MHCII GFP) in phagosomes of primary macrophages from Slamf1+/+ or Slamf1−/− MHC class II–eGFP (B6) mice and 3-μm beads coated with E. coli outer membrane extract. (d) Entry of 3-μm beads coated with E. coli outer membrane extract into lysosomes loaded with Texas red–dextran. Right (c,d), quantification of fluorescence in the microscopy at left. Numbers in bottom right corners (a,c,d) indicate time (in min). Original magnification (a–c), ×60. DIC, differential interference contrast. Data are representative of three combined experiments (a) or at least three independent experiments (b–d) with at least 100 beads or 80 bacteria in each (error bars, s.d.).
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf4

(a,b) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E. coli outer membrane extract; numbers in bottom corners indicate time (in min). α-, anti-. Original magnification, ×60. Right (a,b), quantification of fluorescence. Data are representative of at least three independent experiments (error bars, s.d.).
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf5

(a) Colocalization of SLAM and bacteria in RAW264.7 macrophages transfected with SLAM-mCherry and allowed to phagocytose E. coli–eGFP or S. aureus–eGFP for 0–120 min (time (in min), bottom right corners at left). Original magnification, ×60. Right, quantification of fluorescence at left. Data are representative of two independent experiments with a minimum of 40 bacteria per time point (error bars, s.d.). (b) Immunoblot analysis of phagosome isolates from RAW264.7 macrophages transiently transfected with cDNA encoding Myc-tagged SLAM (Slamf1-Myc) or mock transfected and allowed to phagocytose beads coated with E. coli outer membrane extract for 60 or 120 min. kDa, kilodaltons. Data are representative of at least three independent experiments.
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf6

(a) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top). (b) Luciferase activity in Jurkat cells transfected with the fusion in a (Slamf1-CD3ζ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ (ΔIgV-Slamf1–CD3ζ), and luciferase reporters as in a, then exposed to heat-killed E. coli F18. (c) Luciferase activity in Jurkat cells transfected as in a, then left uninoculated (−) or inoculated with 10 × 108 E. coli F18 (+), followed by the addition of monoclonal antibody (mAb; amount, under graph) 9D1 to SLAM (α-SLAM) or rat immunoglobulin G2b isotype-matched control antibody (Rat IgG2b). (d) Luciferase activity in Jurkat cells transfected as in a, then exposed to medium alone (Med), wild-type E. coli (JM101), or JM101 E. coli mutants lacking both Omp C and Omp F (HN705) or lacking either OmpC (ΔOmpC) or OmpF (ΔOmpF). Data are representative of at least three independent experiments (error bars (a), s.d.).
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf7

(a,b) Production of phagosomal PtdIns(3)P in E. coli–containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX–eGFP) and treated with DsRed-expressing E. coli (E. coli–DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left. (c) HPLC analysis of the production of PtdIns(3)P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract. (d) PtdIns(3)P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages (2010)
figf8

(a,b) Immunoassay of 293 cells transfected with various combinations of full-length SLAM (a) or hemagglutinin-tagged (–HA) tail-less SLAM (b), EAT-2A, V5-tagged (–V5) Vps34-Vps15, and beclin-1; proteins immunoprecipitated (IP) from lysates with monoclonal antibody to SLAM, as well as whole-cell lysates (WCL), were analyzed by immunoblot (IB) with anti-beclin-1, anti-Vps34, anti-V5, anti-hemagglutinin, anti-SLAM or anti-EAT2. (c) Microscopy of RAW264.7 cells transiently transfected with cDNA encoding GFP-tagged beclin-1 and SLAM-mCherry, then treated with beads coated with E. coli outer membrane extract (at a ratio of 10:1, beads/cells) and fixed after 60 min. Data are representative of six (a) or two (b,c) independent experiments.
Modulation of Yorkie activity by alternative splicing is required for developmental stability (2020)
Figure 1

Figure 1. Identification of yki as a B52-regulated alternative splicing target.

(A) Number of genes identified by MAJIQ that display 20% or more variation in splicing between wild-type and B52 depleted cells, in each dataset. We identified 108 shared alternative splicing events, corresponding to 105 genes. A comprehensive list of the 108 events is given in Appendix Table S1.

(B) High confidence protein-protein complexes identified by MIST (Molecular Interaction Search Tool) within the 105 genes identified in (A). Interactions between proteins can be direct or indirect within the complexes.

(C) GO term enrichment analysis of the 105 genes identified in a, performed by PANTHER with Bonferroni correction for multiple testing.

(D) Schematic representation of yki locus and its two isoforms: Yki2 (includes exon 3 and contains 2 WW domains) and Yki1 (skips exon 3 and contains 1 WW domain).

(E and F) RT-PCR and western blot showing the effect of RNAi-mediated B52 depletion in S2R+ cells (E) and in larval wing discs (F).
Modulation of Yorkie activity by alternative splicing is required for developmental stability (2020)
Figure 3

Figure 3. Yki1 isoform is a weaker transcriptional activator than Yki2 isoform.

(A) Comparison of Yki isoforms activity in luciferase assays using UAS-luciferase or 2xSd-Luciferase reporters in S2R+ cells. Error bars represent SD, n=3. *** p-value<0.001; **** p-value<0.0001 (unpaired two-tailed t-tests).

(B) Example of immunostaining used to quantify expression of ex-lacZ in wild type or upon Yki1 or Yki2 overexpression in wing disc posterior domain. GFP labels the posterior domain. Scale bars: 50 µm.

(C) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain (hh-Gal4 driver). For each target gene, relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti-ßgal antibody (for ex-lacZ and diap1-lacZ) or direct visualization of GFP (ban-sensor, in this case posterior domain was visualized by immunostaining against V5-tag present in UAS-Yki transgenes). In the scatter dot plot, each symbol (circle, square and triangle) represents a single wing disc, bars represent mean with SEM. ** p-value<0.01; **** p-value<0.0001 (unpaired two-tailed t-tests).
Modulation of Yorkie activity by alternative splicing is required for developmental stability (2020)
Figure 4

Figure 4. Yki1 isoform lacks some bridging activity and can compete with Yki2 isoform in the nucleus.

(A) Principle of dual-luciferase co-IP. In all experiments IP is performed against the Flag-tag fused to the Firefly Luciferase (FFL). FFL (unfused to Yki) is used as reference to normalize each interaction. The prey protein is fused to Renilla Luciferase (RL) in C-term for most proteins or in N-term (for Wbp2 and Mor).

(B) Drawing of the monitored protein-protein interactions. Interactions previously shown to involve WW domains are indicated by orange arrows, whereas other interactions are schematized by blue arrows.

(C) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly).

(D) Interaction between Sd and Tgi in absence or presence of Yki isoforms.

(E) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms.

(F) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms. In C, D, E and F, bars represent mean and error bars represent SD, n=4. p-values: *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed t-tests).

(G) Luciferase assay using 3xSd-Luciferase reporter in S2 cells, transfected with single or combination of Yki isoforms. Error bars represent SD, n=6. * p-value<0.05; **** p-value<0.0001 (unpaired two-tailed t-tests).

(H) Co-overexpression of Yki isoforms using GMR-Gal4 driver provides evidence of competition between Yki isoforms.
Modulation of Yorkie activity by alternative splicing is required for developmental stability (2020)
Figure 5

Figure 5. Absence of yki alternative splicing reduces the phenotype of B52 depletion in the wing and favors clonal growth.

(A) Structure of the yki2-only allele compared to wild type yki locus. Introns surrounding exon 3 are removed. This allele does not contain any exogenous sequence.

(B) Western blot of adult males and females showing disappearance of Yki1 isoform in the two yki2-only fly lines.

(C) Comparison of posterior/total area ratio of females wings upon depletion of B52 in a yki+ (wild type) or yki2-only background. Each point corresponds to a single wing, bars represent mean with SD (unpaired two-tailed t-tests: **** p-value <0.0001).

(D) Analysis of embryonic lethality according to maternal and paternal genotypes. Bars represent mean and error bars represent SD. Numbers of counted embryos are indicated in each column. Unpaired two-tailed t-tests: **** p-value <0.0001.

(E and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.
Human TLR8 senses UR/URR-motifs in RNA derived from bacteria and mitochondria (2015)
Figure 1

Figure 1 – Specific bacteria and mitochondria ribosomal RNA segments and fractions induce proinflammatory but not type I IFN cytokine production through an endosomal TLR

A Cytokine release by PBMCs challenged with heat inactivated S. aureus (hiSa), and/or RNase A (RA) treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **, p ≤ 0.01; n = 3).

B Alignment of Sa19 and mitochondrial 16S rRNA Sa19 “like” segment (mt, mitochondrial; PTL, peptidyl transferase loop; D, domain; _, transition region) sequence with common core motif (blue) and uracils (red U); *, conserved in human (Hs), cattle (Bt), mouse and rat; G/A underlined, mutated core motif; ma, 6N methylation of adenosine 7.

C Cytokine release of PBMCs transfected with ORNs including TLR7/8 ligand RNA40 (n = 3).

D, E Activity of PBMCs upon bacterial RNA challenge, pretreatment with chloroquine in (D) only (S, Svedberg; r, ribosomal; tot, total; n = 3).

F Activity of PBMCs upon mitochondrial (mt) RNA challenge (Hs, human; triangle, increasing doses; LFA, Lipofectamine 2000; n = 3).

G Activity of PBMCs challenged with ORNs and total S. aureus (Sa) RNA (Myd88d/d, mutant MyD88 expression not impairing LPS driven IL-8 production; n = 2).

H Responsiveness of undifferentiated (undiff) and 3- or 8 days PMA differentiated (ddi) THP1 cells to Sa19 challenge (n = 3).

I Activity of parental and Unc93b1-/--3ddiTHP-1 cells challenged with ORNs (n = 3).

(A-I) Graphs show mean ± SD; -, unchallenged; n.d., not detected; P3C, Pam3CSK4; Loxo, loxoribine.
Human TLR8 senses UR/URR-motifs in RNA derived from bacteria and mitochondria (2015)
Figure 2

Figure 2 – Transcriptome as well as gain and loss of function analyses implicate TLR8 as bacteria and mitochondria ribosomal RNA sensor.

A Cytokine release of 3 days differentiated (ddi) THP1 cells toward stimulation with TLR ligands and bacterial RNAs (S, Svedberg; r, ribosomal; tot, total; n = 3).

B, C Responsiveness of Unc93b1-/--3ddiTHP1 cells treated with T2.5 and challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; n=3).

D Ratios of constitutive mRNA amounts in 3- versus 8ddi (grey bar) and of each of both versus undifferentiated THP1 cells (black or white bar, respectively) according to a transcriptome profiling result (n = 1).

E, F NF-B driven relative luciferase activity (Rel. luc. act.) of hTLR8 overexpressing HEK293 and murine RAW264.7 cells, respectively, upon challenges (vector, empty plasmid; n = 3).

G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness (n = 3).

A-G Graphs show mean ± SD; *, p ≤ 0.05; **, p ≤ 0.01; -, unchallenged; P3C, Pam3CSK4; n.d., not detected; Sa, S. aureus; Ec, E. coli.
Human TLR8 senses UR/URR-motifs in RNA derived from bacteria and mitochondria (2015)
Figure 3

Figure 3 – TLR8 recognizes UR/URR motif containing RNA.

A, B Cytokine release of 3 days differentiated (ddi) THP1 cells challenged with (A) ORNs or (B) bacterial RNA fractions (tot, total; S, Svedberg; r, ribosomal).

C Responsiveness of Tlr8-/--3ddiTHP1 cells challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; *, p ≤ 0.05; **, p ≤ 0.01) upon TLR2 blockade (T2.5).

D Responsiveness of 3ddiTHP1 cells upon challenge with human (Hs) mitochondrial (mt) RNA (triangle, increasing doses).

E-H NF-B driven relative luciferase activity (Rel. luc. act.) of hTLR8+ HEK293 cells or cytokine release by PBMCs all transfected with RNAs indicated and additional uridine (U) if indicated (n.p., not performed; vector, empty plasmid).

A-H Graphs show mean ± SD; each n = 3; -, unchallenged; P3C, Pam3CSK4; Sa, S. aureus; Ec, E. coli; n.d., not detected.