Journal: Cell Cycle

Article Title: TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

doi: 10.4161/cc.23406

Figure Lengend Snippet: Figure 2. Chemotherapy-induced tumor injury enriches for CD44+/MyD88+ EOC stem cells. (A) Paclitaxel treatment (20 mg/kg q3d) enhances growth of tumor implants. (B) Enrichment of CD44 positive cells in tumors from mice treated with Paclitaxel. Tumor implants treated as in (A) were dissociated at the end of the treatment and the number of CD44+ cells was determined by flow cytometry. Note the significant increase in CD44+ cells in the Paclitaxel-treated group.

Article Snippet: Mouse anti-human MyD88, rabbit anti-human β-catenin, rabbit anti-human Oct4 antibodies were purchased from Cell Signaling Technology.

Techniques: Flow Cytometry

Journal: Cell Cycle

Article Title: TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

doi: 10.4161/cc.23406

Figure Lengend Snippet: Figure 3. CD44+/MyD88+ EOC stem cells repair in vitro wound. (A) CD44+/MyD88+ EOC stem cells (i) and CD44-/MyD88- EOC cells (ii), were grown to confluence and “wounded” as described in the “Materials and Methods” section. Note that only CD44+/MyD88+ EOC stem cells repair in vitro wound. (B) GFP+/CD44+/MyD88+ EOC stem cells and RFP+/ CD44-/MyD88- EOC cells were grown to confluence and wounded (i). Wound healing was determined by fluorescent microscope at 24 h (ii), 48 h (iii) and 72 h (iv). Note that only GFP+/CD44+/MyD88+ EOC stem cells can repair the wound. (C) Immunohistochemistry for Ki67 (green staining) was determined in (i) no wound control, (ii) wound edge 4 h after wounding, (iii) wound edge 24 h after wounding and (iv) in wound back 24h after wounding. w, wound area. Note: for wound edge and wound back definitions, please see text.

Article Snippet: Mouse anti-human MyD88, rabbit anti-human β-catenin, rabbit anti-human Oct4 antibodies were purchased from Cell Signaling Technology.

Techniques: In Vitro, Microscopy, Immunohistochemistry, Staining

Journal: Cell Cycle

Article Title: TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

doi: 10.4161/cc.23406

Figure Lengend Snippet: Figure 4. In vitro wound/repair process is associated with CD44+/MyD88+ EOC stem cell self-renewal. (A) Levels of CD44, Nanog, Sox2 and Oct4 were determined by qRT-PCR in control cultures (no wound) and in cells from wound edge (WE) 24h after wounding. (B) Levels of CD44, Oct-4, β-catenin and GAPDH were determined by western blot analysis from control cultures (no wound) and from cells isolated from WE and wound back areas. C, control no wound; WE, wound edge; WB, wound back.

Article Snippet: Mouse anti-human MyD88, rabbit anti-human β-catenin, rabbit anti-human Oct4 antibodies were purchased from Cell Signaling Technology.

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Isolation

Journal: Cell Cycle

Article Title: TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

doi: 10.4161/cc.23406

Figure Lengend Snippet: Figure 5. TLR2 signaling enhances repair and self-renewal. (A) qRT-PCR analyses for TLR2 expression was performed on cancer cells dissociated from the excised tumor fragments at day 24 post-surgery. *p < 0.05. (B) qRT-PCR analyses for TLR2 expression was performed on cancer cells dissociated from tumors of animals treated with Paclitaxel as described in Figure 2A. (C) Levels of TLR2, TLR3 and TLR4 were determined by qRT-PCR from control cultures (no wound) and from cells isolated from wound edge (WE) area 8 h post-wounding. Data represent the average from three independent experiments, measured in triplicate and normalized to GAPDH mRNA. (D) Wounded CD44+/MyD88+ EOC stem cultures were left untreated or treated with 2 µg/ml of PGN or 10 μg/ml of LPS and effect on wound repair was determined. (E) Levels of CD44 and Nanog were determined by qRT-PCR from wounded CD44+/MyD88+ EOC stem cells in the presence or absence of PGN.

Article Snippet: Mouse anti-human MyD88, rabbit anti-human β-catenin, rabbit anti-human Oct4 antibodies were purchased from Cell Signaling Technology.

Techniques: Quantitative RT-PCR, Expressing, Isolation

Journal: Cell Cycle

Article Title: TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

doi: 10.4161/cc.23406

Figure Lengend Snippet: Figure 6. Inhibition of TLR2 or MyD88 activity inhibits repair. CD44+/MyD88+ EOC stem cells were transfected with a dominant-negative form of TLR2 or a dominant-negative form MyD88 prior to wounding. The effect of blocking TLR2 or MyD88 function on repair is then determined. Note the significant decrease on wound repair in cells transfected with TLR2 or MyD88 dominant-negative plasmids. TLR2-dn, TLR2-dominant-negative; MyD88-dn, MyD88-dominant-negative. *p < 0.005.

Article Snippet: Mouse anti-human MyD88, rabbit anti-human β-catenin, rabbit anti-human Oct4 antibodies were purchased from Cell Signaling Technology.

Techniques: Inhibition, Activity Assay, Transfection, Dominant Negative Mutation, Blocking Assay

Journal: Cell Cycle

Article Title: TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

doi: 10.4161/cc.23406

Figure Lengend Snippet: Figure 8. Inhibition of NFκB inhibits repair and self-renewal. (A) CD44+/MyD88+ EOC stem cells were wounded and repair process determined in the presence of absence of the NFκB inhibitor, BAY 11-7082 (2.5 μM). (B) CD44, Sox2, Nanog expression levels were determined using qRT-PCR from wounded CD44+/MyD88+ EOC stem cells in the presence or absence of BAY 11-7082. *p < 0.05.

Article Snippet: Mouse anti-human MyD88, rabbit anti-human β-catenin, rabbit anti-human Oct4 antibodies were purchased from Cell Signaling Technology.

Techniques: Inhibition, Expressing, Quantitative RT-PCR

Journal: NPJ Vaccines

Article Title: IL-1 delineates squalene-based adjuvant efficacy and reactogenicity in a cell-type-specific manner

doi: 10.1038/s41541-026-01420-0

Figure Lengend Snippet: A Study schedule. Mice were injected IM with Ag mixed with A-910823 on days 0 and 14 ( n = 4–10/group), using Myd88 ( B ), Il1r1 ( C ), and Il1rn KO mice ( D ), as well as Myd88 flox/flox (f/f) and Cd11c cre ( E ) or LysM cre mice ( F ). B – F Serum titers of neutralizing antibodies against SARS-CoV-2 on day 28. Bars represent geometric mean titer (GMT); error bars indicate 95% confidence intervals; circles represent neutralizing antibody titers in individual mice. Percentages of T follicular helper (Tfh; PD1 + CXCR5 + ) cells in TCRb + CD4 + cells and percentages of germinal center B (GCB; FAS + GL7 + ) cells in CD19 + cells in draining LNs on day 28. Bars represent means; error bars indicate standard error of the mean; circles represent percentages of Tfh and GCB cells in individual mice. Injection-site swelling scores on day 15. Bars represent means; error bars indicate standard error of the mean; circles represent swelling scores in individual mice. Average body temperature was assessed on Day 12 (08:00–19:59) and on Day 15 (08:00–19:59). Vaccination at day 14 was performed at 18:00. Bars represent means; error bars indicate standard error of the mean. The data were derived from independent experiments: (1) Neutralizing titer/Tfh/GCB measured in the same cohort, (2) Swelling measured in an independent cohort, and (3) Fever measured in an independent cohort. Statistical significance determined using two-tailed Student’s t -test for two-group comparisons or two-way ANOVA. ns, P ≥ 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Il1rn KO mice, Myd88 flox mice, Cd11c cre transgenic mice, and LysM cre transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Injection, Derivative Assay, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis

doi: 10.1186/s12964-024-01930-1

Figure Lengend Snippet: MyD88 D162E mutant mice are protected from DSS-induced acute colitis. Schematic presentation of the DSS-induced colitis experiment. To induce acute colitis, mice were drinking tap water, supplemented with 2% DSS for 8 consecutive days ad libitum. A Disease activity index was defined by observing the stool consistency and the presence of the gross or occult blood. The weight loss was calculated from the weight difference on day1 and day 8 and the colon length was measured from the body of the caecum to the distal part of the rectum. B The part of the colon was washed and IL-6 cytokine was measured using ELISA assay. C Blood was collected and IL-6 was detected in serum using ELISA. D-F Il6 , Il1b , and MyD88 mRNA expression was detected using qPCR in colon tissue homogenate. G MyD88 protein detection from obtained colon tissue of treated mice, using Western blot. β-actin was used as loading control. H Schematic presentation of the experiment. LPS was i.p. injected. After 4 h IL-6 and IL-10 cytokines were analyzed in the serum. Combined data from one ( B , C . H ) or two ( A , D - F ) indep. exp. are shown as mean ± SEM. LN transformation was applied for the data used in statistical tests ( D - F ). Studentʾs unpaired t -test was used. p values of < 0.05 (*), < 0.01 (**) are indicated

Article Snippet: To determine the role of caspase-1 in MyD88 signaling regulation cells were transfected with pcDNA3.1 mouse MyD88 wt (a gift from R. Medzhitov (Addgene plasmid #13,092; http://n2t.net/addgene:13092 ; RRID:Addgene_13092)) or pcDNA3.1 mouse MyD88 D162E plasmids (both 0.05 ng/well) w/o increasing concentrations of pCMV mouse Caspase-1 (0.05, 0.1 ng/well) as well as pELAM1-luciferase (NF-κB promotor; 30 ng/well)) (a gift from C. Kirschning, Germany) and phRL-TK Renilla luciferase (3 ng/well) (Promega) for normalization using lipofectamine 2000 transfection reagent (Invitrogen).

Techniques: Mutagenesis, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Injection, Transformation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis

doi: 10.1186/s12964-024-01930-1

Figure Lengend Snippet: MyD88 protein levels are lower in MyD88 D162E BMDMs, but cells express similar cytokine levels. A MyD88 D162E and MyD88 wt BMDMs were seeded and left untreated for 4 h for MyD88 mRNA detection using qPCR. B BMDMs were seeded and incubated with either cycloheximide (100 μM) or lactacystin (50 μM) for 5 h to inhibit protein translation and proteasomal degradation. Cells were lysed and MyD88 protein was detected using WB. β-actin was used as loading control. BMDMs were stimulated with either LPS or Pam2CSK4 (both 10 ng/ml) or left untreated for 4 h for Il6 and Il10 mRNA detection by qPCR ( C ) and 16 h for IL-6 and IL-10 detection using ELISA ( D ). E Cell tolerance was induced by stimulating BMDMs with LPS (100 ng/ml) for 24 h. Media was changed and after 2 h of resting, cells were restimulated with LPS (10 ng/ml) for 6 h. TNFα was detected using ELISA. A representative experiment of two indep. exp. is shown ( B ). Combined means from four ( C ) and three ( A , D , E ) indep. exp. are shown as mean ± SEM. LN transformation was applied for the data used in statistical tests ( A ). Studentʾs unpaired t -test was used. n.s.- not significant

Article Snippet: To determine the role of caspase-1 in MyD88 signaling regulation cells were transfected with pcDNA3.1 mouse MyD88 wt (a gift from R. Medzhitov (Addgene plasmid #13,092; http://n2t.net/addgene:13092 ; RRID:Addgene_13092)) or pcDNA3.1 mouse MyD88 D162E plasmids (both 0.05 ng/well) w/o increasing concentrations of pCMV mouse Caspase-1 (0.05, 0.1 ng/well) as well as pELAM1-luciferase (NF-κB promotor; 30 ng/well)) (a gift from C. Kirschning, Germany) and phRL-TK Renilla luciferase (3 ng/well) (Promega) for normalization using lipofectamine 2000 transfection reagent (Invitrogen).

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis

doi: 10.1186/s12964-024-01930-1

Figure Lengend Snippet: MyD88 D162E BMDMs experience oxidative stress and have lower glycolysis rates. MyD88 D162E and MyD88 wt BMDMs were seeded. The next day, the cells were stained with CM-H2DCFDA for intracellular ROS detection ( A ), MitoSox Red for mtROS ( B ), MitoTracker Green and MitoTracker Deep Red for dysfunctional mitochondria detection ( C ) and analyzed using flow cytometry. D TEM analysis showing mitochondria of wt BMDMs with well-developed cristae, while MyD88 D162E BMDMs have mitochondria with less cristae (arrows) or without cristae (stars). Bars: 500 nm. E BMDMs were seeded and the next day mito-stress test was performed on Seahorse analyzer with oligomycin (2 μM), FCCP (4 μM) and rotenone (0.5 μM) + antimycin A (0.5 μM). OCR and ECAR were normalized to protein content. F MyD88 D162E and MyD88 wt BMDMs were seeded and primed with LPS (10 ng/ml) or left untreated. Medium was changed and cells were further stimulated with 5 μM nigericin (after 4 h of LPS) for 1 h or 3% DSS (after 2 h of LPS) for 24 h. Secretion of IL-1β and IL-18 was measured using ELISA. BMDMs were stimulated with LPS and Pam2CSK4 (both 10 ng/ml) or left untreated for 4 h and next Il1b mRNA was detected using qPCR. G BMDMs were stimulated with LPS (100 ng/ml) or left untreated for 6 h and expression of NLRP3, proCasp-1, ASC and p62 protein was detected using WB. Gapdh and β-actin were used as loading controls. H , I BMDMs were stimulated with LPS and Pam2CSK4 (both 10 ng/ml) or left untreated for 4 h and A20/Tnfaip3 and p62 mRNA were detected using qPCR. J p62 mRNA expression was detected using qPCR in colon tissue from mice DSS-colitis experiment. Colon tissue was homogenized and lysed, next the p62 protein was detected using WB. β-actin was used as loading control again. Combined means from three (C, F, H) and four ( A , B , E , I ) indep. exp. are shown as mean ± SEM. A representative experiment of two independant exp. is shown ( G ). Combined data from two ( J ) indep. exp. are shown as mean ± SEM. p values of < 0.05 (*) are indicated; n.s.- not significant. Paired t -test was used for cytometry data. LN transformation was applied for the data used in statistical tests ( F , H ). Studentʾs unpaired t -test was used. p values of < 0.05 (*), are indicated. n.s.- not significant

Article Snippet: To determine the role of caspase-1 in MyD88 signaling regulation cells were transfected with pcDNA3.1 mouse MyD88 wt (a gift from R. Medzhitov (Addgene plasmid #13,092; http://n2t.net/addgene:13092 ; RRID:Addgene_13092)) or pcDNA3.1 mouse MyD88 D162E plasmids (both 0.05 ng/well) w/o increasing concentrations of pCMV mouse Caspase-1 (0.05, 0.1 ng/well) as well as pELAM1-luciferase (NF-κB promotor; 30 ng/well)) (a gift from C. Kirschning, Germany) and phRL-TK Renilla luciferase (3 ng/well) (Promega) for normalization using lipofectamine 2000 transfection reagent (Invitrogen).

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Control, Cytometry, Transformation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis

doi: 10.1186/s12964-024-01930-1

Figure Lengend Snippet: MyD88 D162E BMDMs are committed to higher apoptosis rates, but LPS stimulation mitigates preterm apoptosis. A MyD88 D162E and MyD88 wt BMDMs were seeded. After 16 h the cells were stained with annexin V-eFluor450 to determine the apoptosis by flow cytometry. B , C BMDMs were stimulated with LPS (100 ng/ml) or left untreated for 6 h and expression of Bcl-xL and BTK/pBTK was detected using WB. β-actin was used as loading control. BMDMs were seeded and after one hour treated with ibrutinib (4 μM) or left untreated ( D ) or stimulated with LPS (100 ng/ml) or left untreated ( F ). At the same time, caspase-3/-7 green dye was added. Caspase-3/-7 activity was measured for 20 h using a 45-min cycle. E BMDMs were seeded and after one hour, nuclear fractions were isolated and p65 (NF-κB) was detected using WB. PCNA was used as loading control. BMDMs were seeded and stimulated with LPS (100 ng/ml) or left untreated. After 16 h the cells were stained with annexin V-eFluor450 for apoptosis ( G ) or MitoSox Red for mtROS, MitoTracker Green and MitoTracker Deep Red for dysfunctional mitochondria detection, and CM-H2DCFDA for cytosolic ROS detection analyzed via flow cytometry ( H ). Combined means from three ( A , G , H ) indep. exp. are shown as mean ± SEM. Representative data from two ( B , C , E ) or three ( D , F ) indep. exp. are shown. A paired t -test was used for cytometry data. p values of < 0.05 (*) or < 0.01 (**) are indicated

Article Snippet: To determine the role of caspase-1 in MyD88 signaling regulation cells were transfected with pcDNA3.1 mouse MyD88 wt (a gift from R. Medzhitov (Addgene plasmid #13,092; http://n2t.net/addgene:13092 ; RRID:Addgene_13092)) or pcDNA3.1 mouse MyD88 D162E plasmids (both 0.05 ng/well) w/o increasing concentrations of pCMV mouse Caspase-1 (0.05, 0.1 ng/well) as well as pELAM1-luciferase (NF-κB promotor; 30 ng/well)) (a gift from C. Kirschning, Germany) and phRL-TK Renilla luciferase (3 ng/well) (Promega) for normalization using lipofectamine 2000 transfection reagent (Invitrogen).

Techniques: Staining, Flow Cytometry, Expressing, Control, Activity Assay, Isolation, Cytometry

Journal: bioRxiv

Article Title: Macrophage renewal in the small intestine governs early-life control of streptococci

doi: 10.64898/2026.02.11.705249

Figure Lengend Snippet: A. Schematic of the experimental plan. Neonatal mice Myd88 -/- , and Myd88 +/- were infected at P12 with 10 colony forming unit (CFU)/ml GBS via oral gavage. B. Survival rate following GBS colonization at postnatal day 12 in Myd88⁻/⁻ and Myd88⁺/⁻ mice. Myd88 ⁻/⁻ (n= 3), and Myd88 ⁺/⁻ (n= 3). C. Dissemination of GBS into organs (spleen, liver, brain/whole brain) at 2 days post colonization in neonatal mice; Spleen and liver: WT (n= 35), and Myd88 ⁻/⁻ (n= 6), and brain: WT (n= 3), and Myd88 ⁻/⁻ (n= 6) D. Quantification of the GBS Log₁₀ CFU in the blood (per ml) at 2 days post colonization in neonatal mice, WT (n= 25), Myd88⁻/⁻ (n=4). E. Schematic of the experimental plan. Adult mice Myd88 -/- , and Myd88 +/- were infected at P12 with 10 CFU/ml GBS via oral gavage. F. Survival rate following GBS colonized adult mice. WT (n= 9), Myd88⁻/⁻ (n=12), and Myd88⁺/⁻ (n=6). G. Log₁₀ CFU count per gram of feces from Myd88⁻/⁻ and Myd88⁺/⁻ , and WT mice at 7-, 14-, or 21-days post GBS colonization. WT (n=6), Myd88⁻/⁻ (n=4), and Myd88⁺/⁻ (n=6). H. Schematic of the experimental plan. Neonatal Villin cre/+ Myd88 flox/flox mice were infected at P12 with 10 colony forming unit (CFU)/ml GBS via oral gavage. I. Survival rate after GBS colonization at postnatal day 12 in Villin cre/+ Myd88 flox/flox and Villin +/+ Myd88 flox/flox mice. J. Log₁₀ CFU count per gram of feces in Villin cre/+ Myd88 flox/flox (n=8) and Villin +/+ Myd88 flox/flox (n=6) mice at 7-, 14-, or 21-days post GBS colonization. K. Dissemination of the Log₁₀ CFU count GBS into the spleen, liver, and mesenteric lymph nodes in Villin cre/+ Myd88 flox/flox and Villin +/+ Myd88 flox/flox mice, 2 days post colonization at P12. L. Schematic of the experimental plan. Neonatal Lysm cre/+ Myd88 flox/flox mice were infected at P12 with 10 colony forming unit (CFU)/ml GBS via oral gavage. M. Survival rate after GBS colonization at postnatal day 12 in Lysm cre/+ Myd88 flox/flox and Lysm +/+ Myd88 flox/flox mice. N. Log₁₀ CFU count per gram of feces in Lysm cre/+ Myd88 flox/flox (n=7) and Lysm +/+ Myd88 flox/flox (n=10) mice 2 days after GBS colonization at P12, in 2 individual experiments. O. Dissemination of the Log₁₀ CFU count GBS into the spleen, liver, and mesenteric lymph nodes in Lysm cre/+ Myd88 flox/flox and Lysm +/+ Myd88 flox/flox mice, 2 days post colonization at P12.

Article Snippet: LysM Cre/+ Myd88 flox/ flox and Villin Cre/+ Myd88 flox/flox and Myd88 -/- , and Ccr2 -/- mice were obtained from Jackson Laboratories.

Techniques: Infection

Journal: bioRxiv

Article Title: Macrophage renewal in the small intestine governs early-life control of streptococci

doi: 10.64898/2026.02.11.705249

Figure Lengend Snippet: A. Experimental plan for colonization and analysis of Asc -/- at 2 days post-colonization at P14. B. CFU count of the GBS in the blood, Spleen, liver, and brain at 2 days post-colonization at P14. C. Quantitative real-time PCR (qRT-PCR) analysis of Myd88 gene expression in Ly6C - MHC-II⁺ cells isolated from the colon of the Lysm cre/+ Myd88 flox/flox (n=5), and Lysm +/+ Myd88 flox/flox (n=3) mice.

Article Snippet: LysM Cre/+ Myd88 flox/ flox and Villin Cre/+ Myd88 flox/flox and Myd88 -/- , and Ccr2 -/- mice were obtained from Jackson Laboratories.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Isolation

Journal: Nature

Article Title: Secreted fungal virulence effector triggers allergic inflammation via TLR4

doi: 10.1038/s41586-022-05005-4

Figure Lengend Snippet: (a) Silver stained SDS-PAGE gel of recombinant CPL1. n = 5 independent experiments. (b) Arginase-1 FACS on BMDMs stimulated for 24hrs with mock or purified rCPL1 alone (left) or with IL-4 (right). n = 3 biologically independent samples. (c) RNA-seq heatmap of the top 50 BMDM IL-4-induced genes comparing the log 2 fold changes of indicated stimulations to PBS. (d) C. neoformans CFUs after 48hrs of incubation at 37°C, 5% CO 2 in supernatants from BMDMs stimulated for 24hrs as in (c) . n = six biologically independent samples. (e) FACS for IL-4Rα levels on BMDMs stimulated for 24hrs with rCPL1 or mock. (f) Western blot for indicated proteins on BMDMs stimulated as in (c) for 8hrs. Data are representative of three independent experiments. (g) Arginase-1 FACS on STAT3 flox/flox BMDMs transduced with empty vector or iCre retrovirus and stimulated as in (c) . n = five biologically independent samples. (h) Arginase-1 FACS in WT, Tlr2 −/− , Tlr4 −/− , or Tlr2 −/− Tlr4 −/− BMDMs stimulated for 24hrs with rCPL1. n = 3 biologically independent samples. (i) Same as (h) but plus IL-4. n = 3 biologically independent samples. (j) FACS for TLR4 levels on BMDMs stimulated for 1hr with LPS (100ng/ml) or rCPL1. n = 3 biological experiments. (k) Luminescence in HEK293T cells transfected with the indicated plasmids plus NF-kB luciferase and stimulated for 6hrs with either rCPL1 or LPS (100ng/ml). n = 3 biologically independent samples. (l) Arginase-1 FACS in BMDMs stimulated for 24hrs with mock, rCPL1, or rCPL1(Y160A) alone (left) or in combination with IL-4 (right). n = 3 biologically independent samples. Concentrations are IL-4 (10ng/ml) and rCPL1 (111nM) unless otherwise noted. Data are presented as mean values +/− SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Article Snippet: MyD88 −/− , Tlr4 −/− and Stat3 flox/flox mice were purchased from Jackson Labs. Arginase-1-YFP (YARG) reporter mice, IL-4ra −/− and Stat6 −/− mice were a gift from Richard Locksley (UCSF).

Techniques: Staining, SDS Page, Recombinant, Purification, RNA Sequencing, Incubation, Western Blot, Transduction, Plasmid Preparation, Transfection, Luciferase

Journal: Nature

Article Title: Secreted fungal virulence effector triggers allergic inflammation via TLR4

doi: 10.1038/s41586-022-05005-4

Figure Lengend Snippet: (a) Silver stained SDS-PAGE gel of recombinant CPL1. n = 5 independent experiments. (b) Arginase-1 FACS on BMDMs stimulated for 24hrs with mock or purified rCPL1 alone (left) or with IL-4 (right). n = 3 biologically independent samples. (c) RNA-seq heatmap of the top 50 BMDM IL-4-induced genes comparing the log 2 fold changes of indicated stimulations to PBS. (d) C. neoformans CFUs after 48hrs of incubation at 37°C, 5% CO 2 in supernatants from BMDMs stimulated for 24hrs as in (c) . n = six biologically independent samples. (e) FACS for IL-4Rα levels on BMDMs stimulated for 24hrs with rCPL1 or mock. (f) Western blot for indicated proteins on BMDMs stimulated as in (c) for 8hrs. Data are representative of three independent experiments. (g) Arginase-1 FACS on STAT3 flox/flox BMDMs transduced with empty vector or iCre retrovirus and stimulated as in (c) . n = five biologically independent samples. (h) Arginase-1 FACS in WT, Tlr2 −/− , Tlr4 −/− , or Tlr2 −/− Tlr4 −/− BMDMs stimulated for 24hrs with rCPL1. n = 3 biologically independent samples. (i) Same as (h) but plus IL-4. n = 3 biologically independent samples. (j) FACS for TLR4 levels on BMDMs stimulated for 1hr with LPS (100ng/ml) or rCPL1. n = 3 biological experiments. (k) Luminescence in HEK293T cells transfected with the indicated plasmids plus NF-kB luciferase and stimulated for 6hrs with either rCPL1 or LPS (100ng/ml). n = 3 biologically independent samples. (l) Arginase-1 FACS in BMDMs stimulated for 24hrs with mock, rCPL1, or rCPL1(Y160A) alone (left) or in combination with IL-4 (right). n = 3 biologically independent samples. Concentrations are IL-4 (10ng/ml) and rCPL1 (111nM) unless otherwise noted. Data are presented as mean values +/− SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Article Snippet: MyD88 −/− , Tlr4 −/− and Stat3 flox/flox mice were purchased from Jackson Labs. Arginase-1-YFP (YARG) reporter mice, IL-4ra −/− and Stat6 −/− mice were a gift from Richard Locksley (UCSF).

Techniques: Staining, SDS Page, Recombinant, Purification, RNA Sequencing, Incubation, Western Blot, Transduction, Plasmid Preparation, Transfection, Luciferase

Journal: Nature

Article Title: Secreted fungal virulence effector triggers allergic inflammation via TLR4

doi: 10.1038/s41586-022-05005-4

Figure Lengend Snippet: (a) FACS subgating on CD45+YARG+ lung cells from arginase-1-YFP (YARG) mice infected intranasally with 5x10 4 CFU WT C. neoformans for 10 days. (b) Representative histogram of YARG expression in lung interstitial macrophages from mice injected intranasally with either saline or 5x10 4 CFU WT C.n. for 10 days. (c) Quantification of YARG expression by FACS on interstitial macrophages from mice injected intranasally with either saline (N=5 mice), WT (N=11 mice), cpl1Δ (N=14 mice), or qsp1Δ (N=5 mice) Kn99a (5x10 4 CFU) for 10 days; statistical significance determined by one-way ANOVA with Bonferroni test. (d) Kaplan-Meier survival curve analysis of mice infected with WT (N=10mice) or cpl1Δ C.n. (N=10 mice); ****p < 0.0001 by Mantel-Cox test. (e) Representative histogram (left) and quantification of YARG expression on WT, Il4ra −/− , or Stat6 −/− (all N=4 mice) infected for 10 days as in (a) . (f) CFUs from lungs of the indicated mouse genotypes (N=6 mice for each genotype) infected for 10 days with either WT or cpl1Δ strains; statistical significance determined by one-way ANOVA with Bonferroni test. (g) Arginase-1 FACS on lung IMs from WT or Tlr4 −/− mice infected as in (a) ; statistical significance determined by unpaired two-sided T-test. (h) Representative FACS histograms of C.neoformans -mCherry expression in alveolar macrophages (left) or interstitial macrophages (right) after 10 days of infection. (i) Representative FACS histograms of C.neoformans -mCherry expression in interstitial macrophages from YARG mice gated on YARG-negative IMs (left) or YARG-positive IMs (right). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: MyD88 −/− , Tlr4 −/− and Stat3 flox/flox mice were purchased from Jackson Labs. Arginase-1-YFP (YARG) reporter mice, IL-4ra −/− and Stat6 −/− mice were a gift from Richard Locksley (UCSF).

Techniques: Expressing, Infection, Injection, Saline

Journal: Nature

Article Title: Secreted fungal virulence effector triggers allergic inflammation via TLR4

doi: 10.1038/s41586-022-05005-4

Figure Lengend Snippet: (a) Arginase-1 FACS in Tlr4 +/+ or Tlr4 −/− BMDMs stimulated for 24hrs with the indicated concentrations of rCPL1-6xHis purified from C.n. supernatants alone or in combination with IL-4 (10ng/ml) (b). n = three biologically independent samples. Data are presented as mean values +/− SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Article Snippet: MyD88 −/− , Tlr4 −/− and Stat3 flox/flox mice were purchased from Jackson Labs. Arginase-1-YFP (YARG) reporter mice, IL-4ra −/− and Stat6 −/− mice were a gift from Richard Locksley (UCSF).

Techniques: Purification

Journal: Nature

Article Title: Secreted fungal virulence effector triggers allergic inflammation via TLR4

doi: 10.1038/s41586-022-05005-4

Figure Lengend Snippet: (a) Quantification of lung eosinophils in WT or Tlr4 −/− mice infected for 10 days with 5x10 4 CFU C. neoformans . (b) Lung CFUs from Tlr4 +/− or Tlr4 −/− mice infected with wild type C.n. (5x10 4 CFU) for 10 days (c) FACS quantification of lung eosinophils in WT (N=6 mice) or Tlr4 −/− (N=4 mice) mice sensitized intranasally with rCPL1. (d) Model of how secreted CPL1 modulates the macrophage inflammatory state (created using Biorender.com ). p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Article Snippet: MyD88 −/− , Tlr4 −/− and Stat3 flox/flox mice were purchased from Jackson Labs. Arginase-1-YFP (YARG) reporter mice, IL-4ra −/− and Stat6 −/− mice were a gift from Richard Locksley (UCSF).

Techniques: Infection