Journal: bioRxiv

Article Title: Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity

doi: 10.1101/2021.09.29.462388

Figure Lengend Snippet: A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of ISG15, Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

Article Snippet: The following antibodies were used: anti-IL-1β (anti-goat, 1:1000; # AF-401-NA, R&D Systems), anti-caspase-1 (anti-rabbit, 1:1000; #24232, Cell Signaling), anti-AIM2 (anti-rabbit, 1:1000; sc- 515895, Santa Cruz), anti-NLRP3 (anti-mouse, 1:1000; #15101, Cell Signaling), anti-Gasdermin-D (anti-rabbit, 1:1000; #93709, Cell Signaling), anti-Viperin (anti-rabbit, 1:1000 # NBP2-03971, Novus Biologicals), anti-ISG15 (1:1000; #9636, Cell Signaling), anti-phospho-STAT3 (anti-rabbit, 1:1,000; #9145, Cell Signaling), anti- κBα (anti-rabbit, 1:1,000; #4814, Cell Signaling), anti- phospho-Iκ Bα (Ser32) (anti-goat, 1:1,000; #9246, Cell Signaling), anti-phospho-AKT1/2/3 (Ser 473) (anti-rabbit, 1:1000; sc-33437, Santa Cruz), anti-phospho-IKKα/β (Ser176/180)(16A6) (anti- rabbit, 1:1000; #2697,Cell Signaling), anti-phospho-IRF3 (Ser 396) (anti-rabbit, 1:1000; #4947, Cell Signaling), anti-phospho-p-TBK-1/NAK (Ser172) (D52C2) (anti-rabbit, 1:1000; #5483, Cell Signaling), anti-phospho-JNK (anti-rabbit, 1:1000; #9251S, Cell Signaling), anti-phospho-ERK (anti-rabbit, 1:1000; #9101, Cell Signaling), anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9) (anti-rabbit, 1:1000; #4511, Cell Signaling), anti-SARM1 (anti-chicken, 1:70; generated by Icosagen by immunizing chicken with the TIR domain of human SARM1), anti-Flag M2 (1 μ Sigma F3165), anti-HA (1:1000, Santa Cruz Biotechnology sc-805).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Produced, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control

Journal: bioRxiv

Article Title: Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity

doi: 10.1101/2021.09.29.462388

Figure Lengend Snippet: A. il1b, tnfa, il12, cxcl10, ifnb, and isg15 mRNA levels were assessed by qPCR in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. B. il10 mRNA levels were assessed by qPCR in the lungs of infected wild-type (WT), sarm1 -/- , and Sarm1 em1.1Tft mice for 24. C. Percentage of immune cells in the lungs of wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. D. PhenoGraph cluster analysis of immune populations in the lungs wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. E. Heat map showing relative signal intensities of the indicated markers on neutrophils of clusters 13, 15 found in the lungs of infected wild-type mice, and clusters 11 and 13 detected in the lungs of sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. F. Heat map showing relative signal intensities of the indicated markers on alveolar macrophages of clusters 5 and 6 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. G. Heat map showing relative signal intensities of the indicated markers on interstitial macrophages of clusters 16 and 17 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. H. Bacterial load in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. I. Bacterial load in the livers and spleens of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. In panels A, B, H and I values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

Article Snippet: The following antibodies were used: anti-IL-1β (anti-goat, 1:1000; # AF-401-NA, R&D Systems), anti-caspase-1 (anti-rabbit, 1:1000; #24232, Cell Signaling), anti-AIM2 (anti-rabbit, 1:1000; sc- 515895, Santa Cruz), anti-NLRP3 (anti-mouse, 1:1000; #15101, Cell Signaling), anti-Gasdermin-D (anti-rabbit, 1:1000; #93709, Cell Signaling), anti-Viperin (anti-rabbit, 1:1000 # NBP2-03971, Novus Biologicals), anti-ISG15 (1:1000; #9636, Cell Signaling), anti-phospho-STAT3 (anti-rabbit, 1:1,000; #9145, Cell Signaling), anti- κBα (anti-rabbit, 1:1,000; #4814, Cell Signaling), anti- phospho-Iκ Bα (Ser32) (anti-goat, 1:1,000; #9246, Cell Signaling), anti-phospho-AKT1/2/3 (Ser 473) (anti-rabbit, 1:1000; sc-33437, Santa Cruz), anti-phospho-IKKα/β (Ser176/180)(16A6) (anti- rabbit, 1:1000; #2697,Cell Signaling), anti-phospho-IRF3 (Ser 396) (anti-rabbit, 1:1000; #4947, Cell Signaling), anti-phospho-p-TBK-1/NAK (Ser172) (D52C2) (anti-rabbit, 1:1000; #5483, Cell Signaling), anti-phospho-JNK (anti-rabbit, 1:1000; #9251S, Cell Signaling), anti-phospho-ERK (anti-rabbit, 1:1000; #9101, Cell Signaling), anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9) (anti-rabbit, 1:1000; #4511, Cell Signaling), anti-SARM1 (anti-chicken, 1:70; generated by Icosagen by immunizing chicken with the TIR domain of human SARM1), anti-Flag M2 (1 μ Sigma F3165), anti-HA (1:1000, Santa Cruz Biotechnology sc-805).

Techniques: Infection

Journal: Molecular cell

Article Title: Decoding the Function of Expansion Segments in Ribosomes

doi: 10.1016/j.molcel.2018.11.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-14-3-3 γ , Cell Signaling technology , Cat# D15B7, RRID: AB_10827887.

Techniques: Recombinant, SYBR Green Assay, Reporter Assay, Plasmid Preparation, Software, Negative Control

Journal: OncoTargets and therapy

Article Title: FAM163A, a positive regulator of ERK signaling pathway, interacts with 14-3-3β and promotes cell proliferation in squamous cell lung carcinoma

doi: 10.2147/OTT.S214731

Figure Lengend Snippet: FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and anti-14-3-3β antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.

Article Snippet: The HBE, LK2 and SK-MES-1 cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin (BSA) and then incubated with the FAM163A (1:50, sc-390936, Santa Cruz Biotechnology), anti-ERK (#4695, Cell Signaling Technology) and anti-14-3-3β (#9636, Cell Signaling Technology) overnight at 4°C, followed by incubation with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:50, Zhongshan Golden Bridge, Beijing, China) at 37°C.

Techniques: Expressing, Western Blot, Transfection, Stable Transfection, Immunoprecipitation, Staining, Immunofluorescence, Colony Assay