Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: TGFβ blocks IFNα/β release and tumor rejection in spontaneous mammary tumors

doi: 10.1101/336990

Figure Lengend Snippet: (a) Spont-PyMT mice were injected i.p. with DMXAA and then sacrificed after 3 hours and 24 hours to measure mRNA levels of cytokines and chemokines in tumors. The relative expressions compared to DMSO treated mice are shown. Each dot corresponds to one tumor. Cumulative data from CTRL: n= 6 mice and DMXAA: n= 6 mice, from 3 independent experiments, are shown. Tukey’s Multiple Comparison Test. (b and c) The relative expression of these cytokines/chemokines, compared to PBS (CTRL) injected mice, were measured in Spont-PyMT and Trans-PyMT target lesion 3 hours after (b) cGAMP (25μg i.t.) injection (Spont-PyMT mice: n= 3 CTRL and n= 3 cGAMP; Trans-PyMT mice: n= 5 CTRL and n= 13 cGAMP) or (c) LPS (50μg i.p.) injection (Spont-PyMT mice: n= 3 CTRL and n= 3 LPS; Trans-PyMT mice: n= 5 CTRL and n= 10 LPS). In b and c , results are expressed as mean ± s.e.m.

Article Snippet: LPS (1x 50 μg) was injected i.p., cGAMP (InvivoGen, 1 × 25 μg) was injected i.t. and mice were sacrificed 3 hours later.

Techniques: Injection, Comparison, Expressing

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Flow cytometry of live HEK 293T cells labeled with GFP and Alexa Fluor 647 antibodies. Control, untransfected cells (A) or cells transfected with Venus (B) , Astnl-FL-Venus (C) , or Astnl-ΔCTD-Venus , lacking the MACPF, FNIII and ANX-like domains in the C-terminus (D) . The x-axis shows total GFP fluorescence and the y-axis shows surface labeling (Alexa Fluor 647). Thus, cells expressing cytosolic Venus-tagged proteins are indicated in the lower right quadrant (Q3), while double positive cells (GFP+/Alexa Fluor 647+) in the upper right quadrant (Q2) express Venus-tagged proteins exposed on the cell surface. The percentage of cells expressing ASTN1 on the cell surface is depicted. Both ASTN1-FL and ASTNi-ΔCTD were significantly expressed on the cell surface (58 % and 49 % of live transfected cells, respectively).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Flow Cytometry, Labeling, Transfection, Fluorescence, Expressing

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Drosophila S2 cell adhesion assays were prepared in four conditions: Cdh2;GFP + mCherry;GFP (A) , Cdh2;GFP + Astn1;mCherry (B) , Cdh2;GFP + Astn1;mCherry + ASTN1 Fab (C) , and Cdh2-A390;GFP + Astn1;mCherry (D) . ASTN1-positive cells were adhering to the CDH2-expressing aggregates after 30 min (arrows in B1 ), with more co-aggregation seen after 1 h (arrows in B2 ) and 2 h (arrows in B3 ), indicating heterophilic trans interactions. Significantly lower proportions of cells were adhering to the aggregates in the conditions with cells expressing control vector (A) or Astn1;mCherry blocked with ASTN1 Fab fragments (C) . The proportion of mCherry expressing cells in the CDH2;GFP-positive aggregates is quantified in (E) . Expression of CDH2∆390;GFP did not result in cell aggregation within 2 h (D) , demonstrating the importance of the cadherin ectodomain for homophilic and heterophilic interactions and cell adhesion. ** P < 0.01; *** P < 0.001. Scale bar represents 20 μm in (A - D) and 10 μm in inset in (B3).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Expressing, Plasmid Preparation

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) Expression of Cd274 , Ido1 , Ido2 , Cyp1a1 , and Cyp1b1 mRNA was quantified by RT-qPCR in control and CMT167 AhR-KO cells (left two bars in each plot) or after 72 hours of treatment with 10 µM benzo(a)pyrene (B(a)P)(right two bars in each plot). Data from three independent experiments, each in duplicate or triplicate are presented as Gapdh -normalized means + SE. B) Protein extracted from cells treated as in ( A ) was probed by western immunoblotting for PD-L1 and, as a loading control, β-actin. One of three representative western blots is shown on the left and β-actin-normalized protein band densities are on the right. Band density data are presented as means from three experiments, each in triplicate, + SE. C) The percentage of Kyn + CMT167 WT or CMT167 AhR-KO cells treated with vehicle or B(a)P as in ( A ) was quantified by flow cytometry. Data from two experiments, each in triplicate, are presented as means + SE from. *p<0.05, **p<0.01, ****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, Control, Western Blot, Flow Cytometry

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