Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 1. mRNA markers of ER stress. Primary hepatocytes were in- cubated for 12 h with fatty acid depleted BSA, 0.5 mM palmitate or 0.5 mM oleate. A: Xbp-1 mRNA splicing and Chop mRNA expression was determined by RT-PCR. Unspliced (u) and spliced (s) Xbp-1 products are indicated. Tunicamycin treated wild-type cells (Tun) served as a positive control for ER stress. Glyceraldehyde-3-phos- phate dehydrogenase (GAPDH) transcript levels served as internal control for RNA loading. B, C and D: Normalized Chop, ATF3, and ATF6 mRNA levels were measured in samples by quantitative PCR and are shown relative to levels in wild-type cells treated only with BSA. Asterisks represent statistically signifi cant differences between KO and WT: ** P < 0.01. (n = 3 animals/genotype). Error bars in- dicate mean ± SD.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Control, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 2. Protein markers of ER stress. WT (Ncb5or+/+) or KO (Ncb5or/) primary hepatocytes were treated for 12 h with palmitic acid (0.5 mM) or fatty acid free BSA only. In addition WT hepatocytes were treated for 12 h with tunicamycin 1 g/ml. A: BiP, the cleaved N-terminal fragment of ATF6, CHOP, and ATF3 were ana- lyzed by Western blots. B: Top: Western blot determinations of XBP-1. Bottom: Western blot determinations of SCD1 and cytochrome b5. To normalize for protein loading, the same blots were reused for antibody de- tection of -actin.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Western Blot

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 3. Dose response to palmitic acid (Palm) in wild-type and Ncb5or/- cells. A: WT and KO primary hepatocytes were treated with 0, 0.1, 0.25, and 0.5 mM palmitic acid for 12 h. Total RNA was extracted from cells and subjected to RT-PCR for analysis of XBP-1 splicing, Chop and GAPDH mRNA expression. B: ATF3 and C. ATF6 mRNA expression were determined by quantitative PCR. Asterisks represent statistically signifi cant differences between KO and WT: ** P < 0.01. (n = 3 animals/genotype). Error bars indicate mean ± SD.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 6. Sensitivity to tunicamycin. Isolated primary hepatocytes from wild-type mice and NCB5OR null mice were treated for 12 h with increasing concentrations of tunicamycin. After cell col- lection, total RNA was extracted from each sample. Chop, ATF3, and ATF6 mRNA were determined by quantitative PCR. No sig- nifi cant differences were noted between KO and WT cells at each dose of tunicamycin (n = 3 animals/genotype). Error bars indi- cate mean ± SD. that CHOP-mediated apoptosis contributes signifi cantly to -cell loss in B6 Ncb5or / mice.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Isolation, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids

doi: 10.1016/j.jcmgh.2024.101366

Figure Lengend Snippet: Antibodies Used

Article Snippet: ATF3 , Rabbit , Novus, NBP1-85816 , 1:500.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 1. Spontaneous expression of ISGs in ATF3-deficient macrophages. (A–C) WT and Atf32/2 BMDMs analyzed by microarray; fold changes and log2 expression values of the top 50 genes that were expressed more in Atf32/2 than in WT BMDM (A), schematic of microarray analysis and identification of ISGs (B), and an overview of ISGs expressed $2-fold in Atf32/2 BMDMs compared with WT cells (C). (D and E) WT and Atf32/2 BMDMs were analyzed for basal ISG (Irf7, Ch25h, Isg15, and Usp18) or Ifnb1 mRNA expression by qPCR. Graphs show mean 6 SEM combined from four independent experiments. *p , 0.05.

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Expressing, Microarray

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 2. ATF3 reduces PRR-inducible IFN-b ex- pression in mouse macrophages. (A–C and E–G) WT and Atf32/2 BMDMs were stimulated with LPS (100 ng/ml), poly(I:C) (1 mg/ml), or CMA (500 ng/ml) for 4 h and Ifnb1 mRNA was measured by qPCR (A–C) or IFN-b protein by ELISA (E–G). (D, H, and I) WT and Atf32/2

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 4. ATF3 downmodulates IFN-b expression by binding directly to its promoter. (A and B) WT and Atf32/2 BMDMs were stimulated with LPS (100 ng/ml) or poly(I:C) (1 mg/ml) as indicated and p-IRF3, ATF3, total IRF3, and b-actin were measured by immunoblot. Data are repre- sentative of three independent experiments. (C and D) Schematic repre- sentation of the Ifnb1 locus (purple bar represents the TSS and exon). (C) ATF3 ChIP-seq peaks identified in dataset GSE36104 (orange bars); nor- malized ChIP-seq signals in WT (light blue) and Atf32/2 (gray) BMDMs (GSE55317) with significant peaks are indicated by the red bar. Potential ATF3 binding sites are denoted as site 1 and site 2 (light red shading). (D) Visualization of the Ifnb1 locus in activated BMDMs with H3K4me1 (orange), H3K27ac (blue), and PU.1 (green) marks. Gray bars indicate significant peaks. (E and F) HEK293T cells were transfected with Ifnb1 promoter constructs alone (E) or in the presence of ATF3 or control plasmids (F) before 16 h stimulation with 20 ng/well transfected pppRNA (IVT4). Firefly luciferase was measured and fold induction relative to nonstimulated cells is shown. Graphs show mean 6 SEM from three in- dependent experiments. *p , 0.05, **p , 0.01.

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Expressing, Binding Assay, Western Blot, ChIP-sequencing, Transfection, Construct, Control, Luciferase

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 3. Overexpression of ATF3 reduces PRR-mediated IFN-b expression in mouse macrophages. (A–C) Atf32/2 or WT iBMDMs were reconstituted with an empty vector control (ctrl) or with an HA-tagged version of murine ATF3. Atf3 mRNA expression was analyzed by qPCR (A and B) and ATF3, HA, and b-actin protein were analyzed by immunoblot; star indicates a nonspecific band (C). (D and F) Atf32/2 (red shading) or (E and F) WT cells (blue shading) were stimulated with LPS (100 ng/ml) or CMA (500 ng/ml) for 4 h and Ifnb1 or Il12b mRNA was analyzed by qPCR. Data show fold change relative to nonstimulated control cells. Graphs show mean 6 SEM combined from four independent experiments. *p , 0.05, ***p , 0.001.

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 5. ATF3 is a type I IFN-inducible gene in mouse and human immune cells. (A and B) BMDMs were stimulated with IFN-b (1000 U/ml), IFN-a (1000 U/ml), IFN-g (1000 U/ml), or LPS (100 ng/ml) for 10 h before Atf3 mRNAwas measured by qPCR (A) or ATF3 and b-actin protein were analyzed by immunoblot and quantified by densitometry (B). (C) BMDMs were stimulated with IFN-b at indicated doses (U/ml) for 10 h and ATF3 and b-actin protein were analyzed by immunoblot and quantified by densitometry. (D) BMDMs were stimulated with IFN-b (1000 U/ml) as indicated and Atf3 mRNA was measured by qPCR. (E) WT or Ifnar12/2 iBMDMs were stimulated with LPS (100 ng/ml) or IFN-b (1000 U/ml) for indicated times before ATF3 and b-actin protein were measured by immunoblot and quantified by densitometry. (F) Human monocytes from four independent donors were stimulated overnight with IFN-b (10 U/ml) IFN-a (10 ng/ml) or IFN-g (10 ng/ml) before ATF3 mRNA was measured by qPCR. (G–I) Human monocytes, plas- macytoid DCs, or PBMCs from four independent donors were stimulated overnight with IFN-b (1000 U/ml) before ATF3 mRNA was analyzed by qPCR. (J) Human monocytes from four separate donors (D1–4) were stimulated overnight with IFN-b (1000 U/ml) and ATF3 and b-actin protein were measured by immunoblot. Graphs show mean 6 SEM combined from at least three independent experiments (A–E) or four independent donors (F–I). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 6. ATF3 directly regulates a subset of IFN-b–stimulated genes. (A–D) WT and Atf32/2 BMDMs were stimulated with LPS (100 ng/ml) (A and B) or IFN-b (1000 U/ml) (C and D) as indicated and Ch25h mRNA was ana- lyzed by qPCR (A and C) or 25-HC in supernatants measured by mass spec- trometry (B and D). (E) WT and Atf32/2

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Mass Spectrometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATF3 Is a Key Regulator of Macrophage IFN Responses.

doi: 10.4049/jimmunol.1500204

Figure Lengend Snippet: FIGURE 7. ATF3 regulates viral replication in macrophages by modu- lating IFN-b levels. (A) WT and Atf32/2 BMDMs were infected with LCMV at indicated multiplicities of infection (MOI) for 24 h and infected cells were measured for viral replication by flow cytometry. Graphs show average 6 SD of four technical replicates representative of two indepen- dent experiments. (B) Atf32/2 iBMDMs reconstituted with a control (Ctrl) or murine ATF3 were infected with VSV*DG(Luc) as indicated before luciferase activity was measured 6 h postinfection. Graph shows mean 6 SEM combined from four independent experiments. *p , 0.05.

Article Snippet: Generation of mouse macrophages expressing ATF3 A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498,MC201919, OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCTATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCGTATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGGTAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with AscI and NotI.

Techniques: Infection, Cytometry, Control, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors

doi: 10.1038/s41598-018-35806-5

Figure Lengend Snippet: Usp9X inhibition causes Noxa increase and ER stress in MPNST cell lines. Ultrastructural analysis shows features of paraptosis. ( a , b ) ST88-14 cells were transfected for 24 h with either non-targeting (NT)-siRNA or Usp9X-siRNA ( a ) or treated with WP1130 at the concentration of 1.25 and 2.5 µM ( b ). Whole cell extracts were collected prior to Western blot analysis for ATF3, Noxa and ß-actin. Numbers shows protein quantification analyzed through ImageJ. N = 3. ( c – e ) Ultrastructural appearance of untreated control cells using TEM. ( f – h ) After treatment with WP1130 at the concentration of 2.5 µM ( f , g , h ) T265-2c cells showed extensive cytosolic vacuolization (f, red arrows) and swelling of ER (g, red arrowheads) and mitochondria (h, red arrows).

Article Snippet: Primary antibodies were obtained from the following sources: Usp9X (Cell Signaling, Danvers, MA #5751), Mcl-1 (Cell Signaling, Danvers, MA #5453), Noxa (Calbiochem, San Diego, CA #OP180), ATF4 (Cell Signaling, Danvers, MA #11815), ATF3 (Novus Biologicals, Littleton, CO #NBP1-85816), β-actin (Sigma, St. Louis, MO #A5316).

Techniques: Inhibition, Transfection, Concentration Assay, Western Blot, Control

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Rac is dominant over Rho in the initial transcriptome-wide response to ECM stiffness and preferentially represses ATF3. (A,B) Venn diagrams of differentially expressed genes in MEFs cultured with 10% FBS for 1 h on stiff versus soft hydrogels, stiff hydrogels with or without EHT1864, or stiff hydrogels with or without CT04. (C,D) The genes regulated by ECM and Rac in A,B were compared to GO gene lists for transcription factors (TFs), transcription co-regulators (co-reg) and histone modifiers. (E,F) Log 2 (fold change) values and adjusted P -values of the genes regulated by ECM stiffness and Rac and contained within the indicated GO terms above. (G) Serum-starved MEFs were plated on soft or stiff FN-coated hydrogels with 10% FBS for 1 h with DMSO (Ctrl), EHT1864 or CT04. Atf3 mRNA levels were quantified by RT-qPCR. The graph shows mean+s.e.m. ( n =4) with results normalized to the expression level on soft hydrogels. * P <0.05; *** P <0.001 (two-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Dose-dependent effects of ECM stiffness on Rac–GTP, Atf3 mRNA, cyclin D1 mRNA and S phase entry. Serum-starved MEFs were incubated in DMEM containing 10% FBS on FN-coated hydrogels of increasing stiffness (∼2, 8, 15 and 25 kPa). (A) Rac–GTP levels determined at 1 h and graphed relative to Rac activity on the softest hydrogel. Results show mean±s.e.m. ( n =4). (B,C) Atf3 and cyclin D1 mRNA levels determined after 9 h and graphed relative to the mRNA levels on the softest hydrogel. Results show mean±s.e.m. ( n =3). (D) The percentage of EdU-positive nuclei determined at 24 h and graphed relative to EdU incorporation on the softest hydrogel. Results show mean±s.d. ( n =3). Statistical significance for each panel was determined by one-way ANOVA; asterisks show the results of Dunnett's post-tests relative to the softest hydrogel. * P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Incubation, Activity Assay

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: ATF3 repression linked to stiffness-dependent cyclin D1 expression. (A) Serum-starved MEFs on soft or stiff FN-coated hydrogels in DMEM containing 10% FBS were treated with vehicle (DMSO) or EHT1864 for 9 h. Atf3 and cyclin D1 mRNA levels were determined from the same lysates and normalized to mRNA expression levels in cells on soft hydrogels. Results show mean±s.e.m. ( n =3). (B) MEFs were infected with adenoviruses encoding GFP (control) or Rac V12 , serum-starved, and cultured and analyzed as in panel A. Results show mean±s.e.m. ( n =3). (C) MEFs infected with adenoviruses (Ad) encoding GFP (control) or ATF3 were serum-starved, incubated on FN-coated hydrogels with 10% FBS for 15 h, and analyzed by immunoblotting for cyclin D1 and ATF3 with GAPDH as the loading control. (D) Quantification of the immunoblot results in C. The graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal on the soft hydrogels ( n =3). (E,F) Serum-starved ROSA and ATF3 KO MEFs were incubated on stiff FN-coated hydrogels with DMSO (Ctrl) or EHT1864 for 9 h. Lysates were analyzed for the levels of ATF3 and cyclin D1 by immunoblotting. GAPDH was used as the loading control. Panel E shows results from ROSA clone R12 and ATF3 KO clone 1-20, and panel F shows quantification of the combined results from ROSA clones R11, R12 and R15 and ATF3 KO clones 1-20 and 1-29. Data were accrued from four independent experiments, and the graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal in the ROSA control. (G) S phase entry was analyzed by EdU incorporation in ROSA clones (R3, R11, R12 and R15) and ATF3 KO clones (1-20, 1-29, 1-44, 1-48 and 1-49) after serum starvation and incubation on stiff FN-coated hydrogels in DMEM containing 10% FBS for 24 h with DMSO (Ctrl) or EHT1864. Results show mean+s.d. n =7 for the ROSA clones and n =8 for the ATF3 KO clones. (H) Model showing that cyclin D1 is regulated by ECM stiffness and Rac through ATF3. * P <0.05; ** P <0.01; **** P <0.0001 (D, two-tailed unpaired t -tests; F and G, one-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Western Blot, Clone Assay, Two Tailed Test, One-tailed Test

Journal: bioRxiv

Article Title: PBRM1-Dependent PBAF Targeting is Required for EMT and Metastasis in Breast Cancer

doi: 10.1101/2025.10.19.683137

Figure Lengend Snippet: (A) Volcano plot of TF consensus motifs enriched in regions of differential accessibility in NMuMG sg Pbrm1 relative to sgCt cells with 48h TGFβ1 treatment (FDR<0.001). (B) Metagene plots and heatmaps of ChIP-seq enrichment of Fosl2 (left), Fosb (center) and Atf3 (right) in sgCt and sg Pbrm1 cells in both untreated and 48h TGFβ1-treated conditions. Enrichment was plotted for corresponding TF binding sites compiled from all conditions. (C) Venn diagram of Phf10 ChIP-Seq sites with Fosl2 (left), Fosb (center) and Atf3 (right) ChIP-seq sites in sgCt cells with 48h TGFβ1 treatment. Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. To the right of each Venn diagram are the metagene plots and heatmaps of ChIP-seq enrichment of Phf10 with Fosl2 (left), Fosb (center) or Atf3 (right) at the Phf10 (top) and associated TF (bottom) binding sites. (D) Genomic feature distribution of the Fosl2, Fosb, and Atf3 ChIP-seq peaks. (E) Genomic tracks of Phf10 and Atf3 ChIP-seq enrichment in sgCt and sg Pbrm1 cells at Tnfsf13b locus in untreated (top) and 48h TGFβ1-treated (bottom) conditions. (F) Scatter plot of the change in expression for DEGs from sg Pbrm1 vs sgCt with 48h TGFβ1 plotted against DEGs from sh Atf3 vs shScr with 48h TGFβ1. Each data point in the scatter plot represents the log2FC expression value of a single gene in the indicated comparison, x axis : sh Atf3 vs shCt, y axis : sg Pbrm1 vs sgCt. The degree of correlation was calculated using all DEGs. (G) Venn diagram of overlap between DEGs in sg Pbrm1 vs sgCt and DEGs from sh Atf3 vs shScr, both with 48h TGFβ1 treatment. Total number of genes from each condition is indicated in parentheses. The top overrepresented GO terms for the set of genes increased by both sg Pbrm1 and sh Atf3 (top) or decreased by both sg Pbrm1 and sh Atf3 (bottom) were identified using Enrichr pathway analysis. (H) Absolute cell counts of control and sh Atf3 cells with and without 5 ng/mL TGFβ1 treatment. 0.9 million cells were seeded for each cell line and cell counts were taken on day 3 and 6. Representative graph, n=3 biological replicates. Data are represented as mean ± SD. (I) Heatmap representation of the differential accessibility of consensus TF motifs in NMuMG sg Pbrm1 cells with and without TGFβ1 treatment performed using diffTF analysis. Significant differences in weighted means between the groups are shown. (J) Intersection of Phf10 ChIP-seq peaks in NMuMG sgCt cells with Irf1 ChIP-seq peaks in 48h TGFβ1-treated NMuMG cells from a published Irf1 ChIP-Seq dataset (GSE141501). Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. (K) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10 from 48h TGFβ1-treated NMuMG sgCt cells and Irf1 from 48h TGFβ1-treated NMuMG cells from a published dataset at Phf10 and Irf1 shared sites. (L) Genomic tracks of ChIP-seq enrichment of Phf10, Atf3, H3K14ac, and Irf1 in untreated and 48h-TGFβ1 treated NMuMG sgCt at Il15 locus. (M) Intersection of Phf10 ChIP-seq peaks in NMuMG sgCt cells with Snai1 ChIP-seq peaks in pBI3G mouse mesenchymal breast cancer cells from a published dataset (GSE61198). Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. (N) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10 from 48h TGFβ1-treated NMuMG sgCt cells and Snai1 from pBI3G mouse mesenchymal breast cancer cells at Phf10 and Snai1 shared sites. (O) Genomic tracks of ChIP-seq enrichment at Snai2 locus of Phf10, Atf3, and H3K14ac in untreated and 48h-TGFβ1 treated NMuMG sgCt cells and Snai1 ChIP-Seq enrichment in pBI3G mouse mesenchymal breast cancer cells. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001

Article Snippet: Atf3 re-expression was done by transient transfection using the addgene construct# 26115 (pRK-ATF3 was a gift from Yihong Ye) , stable clones were selected by continuous G418 administration (600 μg/mL) and used for experiments.

Techniques: ChIP-sequencing, Binding Assay, Expressing, Comparison, Control

Journal: Brain structure & function

Article Title: Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF

doi: 10.1007/s00429-023-02635-w

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: ATF3 Used for injured motoneuron identification , Recombinant protein corresponding to aa 1-103 in human ATF3 , Mouse/monoclonal , Novus Clone 1685 NBP2-34489 , AB_2786997 Recognizes the epitope: ASAIVPCLSPPGSL (Manufacturer’s information) Not present in uninjured motoneurons , 1:200.

Techniques: Comparison, Expressing, Recombinant