Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) CMT167 Ctrl or CMT167 AhR-KO cells were untreated or treated with 100 ng/ml IFNγ. Cd274 mRNA was quantified by RT-qPCR 24h later. Ido1 , Ido2 , Cyp1a1 , and Cyp1b1 mRNA was quantified 72h later. Data are from three independent experiments, each in duplicate or triplicate, are expressed as fold change of Gapdh -normalized means + SE. B) CMT167 Ctrl or CMT167 AhR-KO cells were left untreated or treated for 24h with 100 ng/ml IFNγ and PD-L1 protein expression assayed by western immunoblotting. A representative immunoblot is on the left and β-actin normalized band densities, averaged from three independent experiments, is on the right. (Bands from IFNγ-treated cells reached saturation prior to bands from untreated cells becoming visible). C) CMT167 Ctrl or CMT167 AhR-KO cells were treated for 24h with IFNγ and IDO1 protein expression assayed by western immunoblotting. A representative immunoblot is on the left and β-actin normalized band densities, averaged from three independent experiments, are on the right. D) CMT167 Ctrl or CMT167 AhR-KO cells were treated with 1-1000 ng/ml IFNγ for 24h and Kyn released into the media quantified via colorimetric assay. Data are averaged from two independent experiments each in quadruplicate + SE. E) CMT167 Ctrl or CMT167 AhR-KO cells were treated with 100 ng/ml IFNγ and Jak2 and Stat1 mRNA quantified 24h later. RT-qPCR data are from three independent experiments, each in triplicate, and presented as Gapdh -normalized means + SE. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Colorimetric Assay

Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) A549 Ctrl or A549 AhR-KO cells were untreated or treated with 100 ng/ml IFNγ for 24h and CD274 , IDO1, and CYP1B1 expression quantified by RT-qPCR. Data from four experiments, each in triplicate, are represented as fold change of GAPDH -normalized means + SE. B) The percent positive PD-L1 + cells treated as in ( A ) was quantified by flow cytometry. Data from three experiments, each in triplicate, are presented as mean percent PD-L1 + + SE. C) The baseline percent of Kyn + A549 ctrl and A549 AhR- KO cells in two experiments, each in triplicate, was determined by flow cytometry. D) A549 Ctrl or A549 AhR-KO cells were treated with 0-1000 ng/ml IFNγ for 24h and Kyn release quantified by the Kyn-specific colorimetric assay using a standard Kyn curve. Data from two experiments, each in quadruplicate, are presented as average μM Kyn + SE. E) A549 Ctrl or A549 AhR-KO cells were left untreated or treated with 100 ng/ml IFNγ for 24h and baseline or 100 ng/ml IFNγ-induced JAK2 , STAT1, and STAT3 expression quantified by RT-qPCR. Data from four experiments, each in triplicate, are presented as average fold change of Gapdh -normalized means + SE. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Colorimetric Assay

Journal: The Journal of biological chemistry

Article Title: Chimeric erythropoietin-interferon gamma receptors reveal differences in functional architecture of intracellular domains for signal transduction.

doi: 10.1074/jbc.272.8.4993

Figure Lengend Snippet: FIG. 7. Phosphorylation of Jak1 and Jak2 kinases. Untreated cells or cells treated with Hu-IFN-g or Epo were lysed and immunoprecipi- tated (I.P.) with monoclonal anti-phosphotyrosine antibodies (anti-P-Tyr panels), polyclonal anti-Jak1 antibodies (anti-Jak1 panels) or anti-Jak2 antibodies (anti-Jak2 panels) as described under “Materials and Methods.” The cell lines are indicated on the figure and are defined in the legend to Fig. 1. Immunoprecipitates were resolved on SDS-PAGE, transferred to PVDF membranes and the blots probed with anti-Jak1 or anti-Jak2 antibodies as noted under the respective horizontal panels.

Article Snippet: Rabbit anti-Jak2 antibody (catalogue no. SC-294) and rabbit anti-Stat5 antibody (catalogue no. SC-835) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, SDS Page

Journal: The Journal of biological chemistry

Article Title: Chimeric erythropoietin-interferon gamma receptors reveal differences in functional architecture of intracellular domains for signal transduction.

doi: 10.1074/jbc.272.8.4993

Figure Lengend Snippet: FIG. 8. Schematic representation of receptor complexes. A represents the IFN-gR1 homodimer bound to IFN-g. The cytoplasmic domains of the two chains are too far apart to permit transactivation of the two Jak1 kinases. B represents the active heteromeric IFN-g receptor com- plex with two IFN-gR1 and two IFN-gR2 subunits per complex. The IFN-g ho- modimer binds to two IFN-gR1 chains, followed by its interaction with two IFN- gR2 chains. The associated Jak2 and Jak1 kinases activate one another by transphosphorylation, with subsequent phosphorylation and dimerization of Stat1a. C depicts the EpoR/gR1 ho- modimer, which, unlike the IFN-gR1 ho- modimer, permits transactivation of the two Jak1 molecules. D illustrates the structure of the heterodimer of EpoR/gR1 and EpoR/gR2, which is the putative ac- tive receptor complex.

Article Snippet: Rabbit anti-Jak2 antibody (catalogue no. SC-294) and rabbit anti-Stat5 antibody (catalogue no. SC-835) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: Multiple sequence alignment of 12 death domains (DDs) from human proteins containing death domain. The red asterisk indicates the conserved arginine site among different DDs, which could be GlcNAcylated by NleB/SseKs. (B) Site distribution of amino acids in 12 death receptor domains containing conserved arginine by Weblogo. (C) Phylogenetic tree of the 12 DDs constructed on the basis of sequence similarity. (D-G) Identification of the physiological substrates of arginine GlcNAc transferase NleB/SseKs during bacterial infection of mammalian cells. 293T cells transfected with pCS2-1Flag-TNFR1 DD, pCS2-1Flag-TRADD DD, pCS2-1Flag-FADD, and pCS2-1Flag-RIPK1 DD were infected with the indicated modified EPEC strains or Salmonella strains. After infection, cells were lysed, and proteins were immunoprecipitated with FLAG M2 beads. Samples were loaded onto SDS-PAGE gels and were immunoblotted with anti-Flag, anti-Arg-GlcNAc, and a loading control anti-tubulin. Data in Fig (D - G) are from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Sequencing, Construct, Infection, Transfection, Modification, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: ( A ) Multiple sequence alignment of 37 death domains (DDs) from human death domain-containing proteins. ( B ) Identification of the physiological substrates of arginine GlcNAc transferase NleB/SseKs during EPEC infection of mammalian cells. 293T cells transfected with pCS2-1Flag-TRADD DD, pCS2-1Flag-FADD DD, and pCS2-1Flag-RIPK1 DD were infected with the indicated modified EPEC strains. After infection, cells were lysed, and proteins were immunoprecipitated with FLAG M2 beads. Samples were loaded onto SDS-PAGE gels and were immunoblotted with anti-Flag, anti-Arg-GlcNAc, and a loading control anti-tubulin. Blot data were derived from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Sequencing, Infection, Transfection, Modification, Immunoprecipitation, SDS Page, Derivative Assay

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: An arginine point mutation screen of hTRADD to investigate its ability to be GlcNAcylated by SseK1. 293T cells were transfected with the indicated plasmid combinations. The samples of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input) were immunoblotted with corresponding antibodies. Anti-tubulin was used as a loading control. (B) Electrospray ionization mass spectrometry (ESI-MS) determination of the total mass of the site-directed TRADD mutants purified from bacteria. GST-TRADD DD, GST-TRADD DD (R235A), GST-TRADD DD (R245A), and GST-TRADD DD (R235A/R245A) were expressed alone (upper panel) or co-expressed with His-SseK1 (lower panel) in E. coli BL21 (DE3) strain. The resulting mass spectra were shown. The resulting mass spectra were shown. The black bar and red bar denote unmodified and GlcNAcylated TRADD DD, respectively. (C) The percentage of site-directed TRADD mutants GlcNAcylated by SseK1. (D) HCD analysis of the peptides of TRADD DD R245 GlcNAcylated by SseK1 in bacteria. The fragmentation patterns of the generated b and y ions were shown along the peptide sequence on the top of the spectrum. (E) Modification of TRADD and TRADD variants by SseK1 upon S. Typhimurine infection. 293T cells was transfected with plasmids carrying TRADD and the site-directed TRADD mutants, and then infected with the indicated Salmonella strains. After 15-hour infection, cells were lysed and proteins were immunoprecipitated with FLAG M2 beads. Samples were loaded onto SDS-PAGE gels, followed by immunoblots with anti-Flag and anti-Arg-GlcNAc antibodies. (F) Identification of the site of TNFR1 GlcNAcylated by SseK3 in bacteria. (G) Summary of ESI-MS determination of the total mass of TNFR1 and its point mutants co-expressed with SseK3 in bacteria. Data in Fig (A, E, and F) are from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Mass Spectrometry, Purification, Generated, Sequencing, Modification, Infection, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: (A) Ectopic expression of NleB/SseKs effectors showed the related subcellular localization and modification pattern in transfected HeLa cells. GFP-NleB, GFP-SseK1, GFP-SseK2, and GFP-SseK3 were expressed ectopically in HeLa cells. In HeLa cells, green indicated immunofluorescence staining of GFP and arginine-GlcNAcylated proteins. Blue indicated DAPI staining of nuclei, and red indicated GM130 staining of the Golgi structure. (B) Analysis of NleB/SseKs auto-arginine-GlcNAcylation by Western blot. Recombinant purified NleB/SseKs and their enzymatic mutants were analyzed on SDS-PAGE gels, followed by immunoblotting with anti-Arg-GlcNAc. (C) ESI-MS analysis determination of the total mass of the NleB/SseKs purified from bacteria. The black bar denotes unmodified protein. For NleB and SseK3, the red bar denotes GlcNAcylated form with 203-Da increase, while for SseK1, the red bar denotes GlcNAcylated (203 Da) and acetylated (42 Da) form with 245-Da increase. (D) Arginine-GlcNAcylation percentage of NleB/SseKs. (E) Summary of ESI-MS determination of the total mass of NleB/SseKs. Data in (A and B) are representative from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Expressing, Modification, Transfection, Immunofluorescence, Staining, Western Blot, Recombinant, Purification, SDS Page

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: Effects of the auto-arginine-GlcNAcylation of NleB on enzyme activity towards death domain protein. The coupled anti-Arg-GlcNAc beads were incubated with 50 μg of purified NleB for enrichment with auto-arginine-glycosylated NleB. Beads enriched with auto-arginine-glycosylated proteins were used in vitro glycosylation assay. Samples were loaded onto SDS-PAGE gels and were immunoblotted with anti-Flag and anti-Arg-GlcNAc. (B) Mass spectrometry analysis of 203-Da increase in the total molecular weight of the auto-arginine-GlcNAcylation site-directed mutant proteins. (C) Effects of the modification site mutation of NleB/SseKs. 293T cells were transfected with the indicated plasmids. After 24-hour transfection, cells were lysed, and proteins were immunoprecipitated with α-FLAG conjugated beads. Samples were loaded onto SDS-PAGE gels, followed by immunoblot with anti-Flag, anti-Arg-GlcNAc, and a loading control anti-tubulin. (D-F) Effects of the auto-arginine-GlcNAcylation on cell death inhibition of NleB and SseKs. HeLa cells infected with the indicated EPEC strains and Salmonella strains were stimulated with TNF-α and TRAIL. Cell viability was determined by measuring ATP levels. Black bars or white bars denoted unstimulated or stimulated, respectively. Data in (A) and (C-F) are representative from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Activity Assay, Incubation, Purification, In Vitro, SDS Page, Mass Spectrometry, Molecular Weight, Mutagenesis, Modification, Transfection, Immunoprecipitation, Western Blot, Inhibition, Infection