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: NPJ breast cancer

Article Title: The human intermediate prolactin receptor is a mammary proto-oncogene.

doi: 10.1038/s41523-021-00243-7

Figure Lengend Snippet: Fig. 5 hPRLrL + I heterodimer signal transduction. MCF10AT transfectants were PRL (250 ng/mL) stimulated for 0, 15, and 30 min, and phospho-IB was utilized to assess the activation status of a, b Jak2/Stat5a and c, d Mek/Erk. Band intensities were quantified using densitometry. Significance was determined by comparing each condition to the MCF10AT-EV negative control for each respective time point. *p < 0.05, n = 3.

Article Snippet: Antibodies used for these studies were obtained from the following sources at the indicated titer: hPRLr ECD (35–9200, Invitrogen, 1:1000), pY-Stat5a (9359S, Cell Signaling, 1:1000), Stat5a (sc-1081, Santa Cruz Biotechnology, 1:1000), pY-Jak2 (3776S, Cell Signaling, 1:500), Jak2 (3230S, Cell Signaling, 1:500), pp44/42 (9101S, Cell Signaling, 1:1000), p44/42 (9102S, Cell Signaling, 1:1000), pS-Mek (9121S, Cell Signaling, 1:1000), Mek (9122S, Cell Signaling, 1:1000), KRAS (14412S, Cell Signaling, 1:1000), hPRLrI (New England Peptide, 1:5000), pS349-hPRLr (Serge Y. Fuchs, M.D., Ph.D., University of Pennsylvania, 1:100), Vinculin (MCA465GA, Bio-Rad, 1:1000).

Techniques: Transduction, Activation Assay, Negative Control

Journal: Frontiers in immunology

Article Title: PARP Inhibitor Upregulates PD-L1 Expression and Provides a New Combination Therapy in Pancreatic Cancer.

doi: 10.3389/fimmu.2021.762989

Figure Lengend Snippet: FIGURE 4 | Pamiparib treatment induces PD-L1 expression via JAK2/STAT3 pathway. (A) Cells were pretreated with pamiparib (100 mM, 12h) and PD-L1 expression was assessed by immunoblotting after treatment with the concentrations (20 mM) of AG490 for 24h. (B) Cells were pretreated with pamiparib (100 mM, 12h) and PD-L1 expression was assessed by protein blotting after treatment with stattic (20 mM) for 24h. (C) Protein expression of phospho-JAK2 (p-JAK2), JAK2, and PD-L1 in SW1990 cells after being treated with pamiparib (100 mM) for the indicated times. (D) Protein expression of phospho-STAT3(p-STAT3), STAT3 and PD-L1 in SW1990 cells after being treated with pamiparib (100 mM) for the indicated times. GAPDH was used as a loading control. (E) IHC staining of p-STAT3 of nude mouse xenograft tumors in control group and treated with pamiparib group. Scale bar, 100 mm. (F) IHC staining score showed the expression of p-STAT3 was significantly upregulated in nude mice pamiparib treated group. Data are mean ± SD; n = 3 samples per group. The IHC results were analyzed by Pearson c2 test. *P < 0.05.

Article Snippet: The primary antibodies are PD-L1 (CST #13684, Cell Signaling Technology), PD-L1 (17952- 1-AP, Santa Cruz), STAT3 (CST #9139, Cell Signaling Technology), phosphorylated STAT3 (CST #9145, Cell Signaling Technology), JAK2 (17670-1-AP, Proteintech), phosphorylated JAK2 (CST #4406, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), phosphorylated AKT (66444-1-Ig, Proteintech), ERK (CST #4696, Cell Signaling Technology), phosphorylated ERK (CST #3510, Cell Signaling Technology), PARP-1 (sc-8007, Santa Cruz), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Control, Immunohistochemistry

Journal: Frontiers in immunology

Article Title: PARP Inhibitor Upregulates PD-L1 Expression and Provides a New Combination Therapy in Pancreatic Cancer.

doi: 10.3389/fimmu.2021.762989

Figure Lengend Snippet: FIGURE 8 | Diagram summarizing that pamiparib treatment induces PD-L1 expression mainly via JAK2/STAT3 in pancreatic cancer (details provided in the Discussion section).

Article Snippet: The primary antibodies are PD-L1 (CST #13684, Cell Signaling Technology), PD-L1 (17952- 1-AP, Santa Cruz), STAT3 (CST #9139, Cell Signaling Technology), phosphorylated STAT3 (CST #9145, Cell Signaling Technology), JAK2 (17670-1-AP, Proteintech), phosphorylated JAK2 (CST #4406, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), phosphorylated AKT (66444-1-Ig, Proteintech), ERK (CST #4696, Cell Signaling Technology), phosphorylated ERK (CST #3510, Cell Signaling Technology), PARP-1 (sc-8007, Santa Cruz), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Expressing

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: Oncology reports

Article Title: Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

doi: 10.3892/or.2017.5588

Figure Lengend Snippet: Figure 2. Silibinin downregulates the expression of Jak2/STAT3 signaling proteins in a dose- and time-dependent manner. (A) Western blot analyses showing the concentration dependent effect of silibinin in MDA‑MB‑231 cells following exposure to silibinin for 24 h. (B) Relative levels of the pSTAT3, STAT3, pJak2, and Jak2 proteins. (C) Time-dependent effect of silibinin on protein expression in MDA‑MB‑231 cells. (D) Relative expression levels of pSTAT3, STAT3, pJak2, and Jak2, measured using densitometry. These data were normalized to actin levels, and then shown as a percentage of the control. The data presented are representative of three independent experiments. Statistical analyses were conducted using the t-test (**p<0.01, ***p<0.001).

Article Snippet: An anti phosphor Jak2 antibody (Tyrosine residue 1007/1008) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Control

Journal: Oncology reports

Article Title: Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

doi: 10.3892/or.2017.5588

Figure Lengend Snippet: Figure 7. Schematic representation of the inhibition of invasive mechanisms provoked by silibinin in MDA‑MB‑231 cells. Silibinin inhibits Jak2 expression and phosphorylation, resulting, in turn, in the inhibition of STAT3 expression, phosphorylation, nuclear translocation, and DNA binding activity. Consequently, STAT3's down-stream targets are inhibited (including MMP2), resulting in reduced cell migration and invasion.

Article Snippet: An anti phosphor Jak2 antibody (Tyrosine residue 1007/1008) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing, Phospho-proteomics, Translocation Assay, Binding Assay, Activity Assay, Migration