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: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 3 SNORD52 mediates M2 macrophage polariza- tion and activates JAK2/ STAT6 pathway. A Following transfection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the mRNA expression of SNORD52 was detected by qRT-PCR. ****P < 0.0001 vs. OE-NC. B Following trans- fection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the protein levels of Arginase-1 and CD163 were determined through Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. OE-NC. C The ratio of CD206-positive or CD163-positive cells following transfection of OE-SNORD52/ OE-NC was analyzed by flow cytometry. ***P < 0.001 vs. OE-NC. D Following transfec- tion of OE-SNORD52/OE-NC in THP-1 and U937 for 48 h, the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. OE-NC. E The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the mRNA expression of SNORD52 was detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ASO-NC. F The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the protein levels of Arginase-1 and CD163 were determined through Western blotting. ***P < 0.001 vs. ASO-NC. G The ratio of CD206-positive or CD163-positive cells following transfection of SNORD52- ASO-1/-2 or ASO-NC was analyzed by flow cytometry. ***P < 0.001 vs. ASO-NC. (H) The IL-4 and IL-13 treated THP-1/ U937 macrophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. ASO-NC. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Incubation

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 5 Huh7 cells-derived exosomal SNORD52 mediates M2 macrophage polarization through activating JAK2/STAT6 pathway. A Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the levels of JAK2/STAT6 pathway-related pro- teins in receptor THP-1 macrophages were measured by Western blotting. B Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the ratio of CD206-positive or CD163-positive THP-1 macrophages was analyzed by flow cytometry. **P < 0.01, ***P < 0.001. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Derivative Assay, Co-Culture Assay, Western Blot, Flow Cytometry

Journal: eLife

Article Title: Single methyl groups can act as toggle switches to specify transmembrane Protein-protein interactions

doi: 10.7554/elife.27701

Figure Lengend Snippet: Figure 3. Biochemical analysis of EPOR activation. (A) Extracts were prepared from BaF3/hEPOR cells (left panels) or BaF3/mEPOR cells (right panels) expressing MSCVp (V), LH5, LH5-L9I (L9I), or LH5-I10L (I10L). MSCVp-expressing cells were also acutely treated with EPO, as indicated. Extracts were immunoprecipitated with anti-HA antibody and immunoblotted with anti-phosphotyrosine antibody PY100 (phospho-EPOR). The multiple species represent minor phosphorylated forms of the EPOR. The same blot was stripped and reprobed for total EPOR by using anti-HA antibody. (B) Cell extracts described in panel A were immunoblotted with anti-phospho-JAK2 antibody (phospho-JAK2). The same blot was stripped and reprobed for total JAK2 using anti-JAK2 antibody. Size of protein markers (in kDa) is shown in A and B. (C) Extracts were prepared from BaF3/hEPOR cells (top panel) or BaF3/mEPOR cells (bottom panel) expressing MSCVp vector (V), FLAG-tagged LI-5 (a traptamer that specifically activates PDGFbR [Heim et al., 2015]), or FLAG-tagged (+) or untagged (-) LH5, LH5-L9I, or LH5-I10L. Extracts were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody. (D) Extracts of BaF3 cells expressing the full-length hEPOR (fl) and/or the D259 truncated hEPOR (t) and the indicated traptamer or MSCVp (V) were immunoprecipitated with anti-C-20 antibody, which recognizes only full-length EPOR, and immunoblotted with anti-HA antibody (C-20 IP) (top panel); or were directly immunoblotted with anti-HA antibody (input EPOR) (bottom panel). – indicates no traptamer. The full-length and truncated EPORs are indicated by the bold and thin arrows, respectively. DOI: https://doi.org/10.7554/eLife.27701.004 The following figure supplements are available for figure 3:

Article Snippet: To detect phosphorylated EPOR, a mouse anti-phosphotyrosine monoclonal antibody PY100 (Cell Signaling) was used at a 1:1000 dilution; to detect phosphorylated JAK2, a rabbit anti-phospho-JAK2 monoclonal antibody (Tyr1008) (clone D4A8, Cell Signaling) was used at 1:1000 dilution; to detect total JAK2, a rabbit anti-JAK2 monoclonal antibody (clone D2E12, Cell Signaling) was used at 1:1000 dilution; to detect HA-tagged EPORs, an HRP-conjugated mouse anti-HA (clone 6E2, Cell Signaling) was used at 1:1000 dilution.

Techniques: Activation Assay, Expressing, Immunoprecipitation, Plasmid Preparation

Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 1 The pedigree of the fam- ily with the JAK2 L604F muta- tion. The patient and her sister have the L604F mutation (blue) in a homozygous state, and her parents have heterozygous L604F. The germline L604F probably arises from a common ancestor pair (encircled in blue). The presence of the mutation in the nontested family members is presumed (light blue) as a highly likely way to yield the results obtained in the tested members

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Mutagenesis

Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 3 Effect of endogenous JAK2 mutations in HeLa cells. Western blot analysis of HeLa cells with V617F or L604F JAK2 mutations introduced by CRISPR. The expected gene modification was confirmed by sequencing. Eight independent harvests were performed for each modified subline and the wild type (WT) parental line. The cell lysates were analyzed in groups – each western-blot membrane contained 4 WT samples and 4 samples from one mutated subline. The measured band intensities were normal- ized to ACTIN and related to the mean value from WT samples included in the given membrane (100%). Top: repre- sentative examples of JAK2 and pJAK2 Tyr1007/1008 signals. ACTIN was used as the loading control. Bottom: means ± SD of the relative band intensities from 8 independent samples for each JAK2 variant. The differences between modified sublines and the WT control were evaluated by unpaired Student´s t test (** p < 0.01, *** p < 0.001)

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Western Blot, CRISPR, Modification, Sequencing, Membrane, Control, Variant Assay