Journal: Epigenetics & Chromatin

Article Title: Aging is associated with highly defined epigenetic changes in the human epidermis

doi: 10.1186/1756-8935-6-36

Figure Lengend Snippet: Characterization of differentially methylated regions (DMRs). (A) Hypermethylated and hypomethylated DMRs show distinct chromatin states. DMRs that become hypermethylated in old epidermis (hyperDMRs) are enriched for H3K4me3 and H3K27me3, while DMRs that become hypomethylated in old epidermis (hypoDMRs) are enriched for H3K27ac. Numbers below the x-axis indicate the distance from the center of the DMR (dashed vertical line) in bp. (B) Enrichment of DMRs within defined genome segments. Bars indicate the ratio of the observed DMR frequency and the average frequency across the genome. Blue bars represent DMRs that are hypomethylated in the old epidermis, red bars represent DMRs that are hypermethylated in the old epidermis. (C) The ERBB receptor feedback inhibitor 1 (ERRFI1) promoter region harbors a representative DMR. ERRFI1 is required for proper epidermal homeostasis and is expressed at lower levels in old epidermis samples (right panel, blue and red bars indicates expression in the young and old epidermis samples, respectively). Blue lines indicate methylation ratios in the young epidermis, red lines indicate methylation ratios in the old epidermis. The green bar indicates the position of the ERRFI1 promoter CpG island. (D) An annotated active enhancer element from the low-density lipoprotein receptor ( LDLR ) gene region harbors a DMR. Age-related methylation changes that were associated with lower expression levels of LDLR in the old sample (right panel, blue and red bars indicates expression in the young and old epidermis samples, respectively), which may promote the formation of Xanthelasma, a dermatological lesion often found in the elderly population.

Article Snippet: The following assays were used, according to the manufacturer’s recommendations: CTGF (Hs01026927_g1), SPRR1A (Hs00954595_s1), SPRR1B (Hs00234164_m1), KRT16 (Hs00373910_g1), KRT17 (Hs01588578_m1), VCL (Hs00247826_m1), HAS3 (Hs00193436_m1), FDZ10 (Hs00273077_s1), NPR3 (Hs00168558_m1), VPS37B (Hs00226582_m1), ERRFI1 (Hs00219060_m1), PLIN2 (Hs00605340_m1), RHPN2 (Hs00369111_m1), RGMA (Hs00297192_m1), PI4KB (Hs01090927_m1).

Techniques: Methylation, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 1 ERRFI1 deficiency ameliorated IR-induced hepatic injury and hepatocellular apoptosis. (A) Schematic diagram showed the hepatocyte-specific ERRFI1 knockout strategy and ischemia reperfusion model. (B, C) Western blot analysis of ERRFI1 protein level in liver tissues from wild type (WT) mice and hepatocyte-specific ERRFI1-knockout (ERRFI1-HKO) mice with sham treatment or ischemia for 90 min followed by reperfusion for 6 h, and quantita tive analysis is shown. (D) Liver function assessed by ALT and AST of mice with different treatment. (E) Liver pathology was determined by H&E staining (scale bar: 100 μm). (F) Suzike’s injury score was used to assess the degree of injury based on H&E staining. (G, H) TUNEL staining of apoptotic cells in liver tissues from WT mice and ERRFI1-HKO mice under different conditions (scale bar: 50 μm), and quantification showing the percentage of apoptotic cells. (I, J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in liver tissues from WT mice and ERRFI1-HKO mice after IR injury. For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 2 ERRFI1 deficiency protected against hepatic IR-induced ferroptosis. (A) Intracellular ROS level was determined by DCFH-DA staining after hepatic IR. (B) Immunohistochemical staining of 8-OHdG in liver tissues (scale bar: 50 μm). (C, D) The content of MDA and the level of GSH in the livers of mice subjected to sham treatment or to an induction of IR. (E) Hepatic Fe2+ content in each group. (F) The mRNA levels of ACSL4, SLC7A11, and GPX4 in liver tissues of mice with different treatments. (G) Representative immunohistochemical images of ACSL4, SLC7A11, and GPX4 in liver tissues (scale bar: 50 μm). For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 3 Knockdown of ERRFI1 inhibited apoptosis of hepatocytes induced by hypoxic-reoxygenation. (A) Expression of ERRFI1 in L-02 cells following hy poxia/reoxygenation (H/R) and ERRFI1 knockdown at the mRNA level was determined by real-time PCR. (B) Cell viability of ERRFI1-silenced L-02 cells after OGD/R exposure. (C, D) Apoptosis-positive cells were detected by TUNEL staining (scale bar: 50 μm). (E, F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in L-02 cells under indicated conditions. For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 4 Knockdown of ERRFI1 suppressed OGD/R-induced ferroptosis in hepatocytes. (A) ROS level in ERRFI1-silenced L-02 cells exposed to hypoxia/ reoxygenation. (B) Flow cytometry analysis of lipid peroxidation using C11-BODIPY 581/591 in L-02 cells under indicated conditions. (C, D) MDA content and GSH level in L-02 cells cultured under indicated conditions. (E) Fe2+ content in ERRFI1-silenced L-02 cells after OGD/R exposure was determined. (F) Real-time PCR showed the mRNA levels of ACSL4, SLC7A11, and GPX4 in response to ERRFI1 knockdown under H/R conditions. (G, H) Fluorescence im munostaining of ACSL4, SLC7A11, and GPX4 in L-02 cells transfected with sh-ERRFI1 during H/R injury (scale bar: 20 μm). For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knockdown, Flow Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence, Transfection

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 5 ERRFI1 directly interacted with GRB2 and maintained its stability by hindering its proteasomal degradation. (A-C) Transcript and protein levels of GRB2 in ERRFI1-silenced L-02 cells were detected. (D) Co-immunoprecipitation of ERRFI1 and GRB2. L-02 cells were subjected to GRB2 immunoprecipita tion and subsequent immunoblotting of ERRFI1 and GRB2. (E, F) L-02 cells were transfected with sh-ERRFI1 and treated with CHX for the indicated times. Western blot analysis showed the expression of GRB2. (G, H) The expression of GRB2 in sh-ERRFI1-transfected L-02 cells with or without MG132 treatment. For statistical analysis, Student’s t test was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 6 ERRFI1 facilitated OGD/R-induced injury of hepatocytes in a GRB2-dependent manner. (A-D) L-02 cells were transfected with GRB2 overexpression plasmid or vector plasmid, followed by 4 h of hypoxia and 12 h of reoxygenation. Cell apoptosis was detected by TUNEL staining. Scale bar: 20 μm (A, B). Lipid peroxidation was measured using C11-BODIPY 581/591 (C). Fe2+ content was determined (D). (E, F) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 20 µM Z-VAD-FMK (ZVF, an apoptosis inhibitor). L-02 cells were treated with 5 µM camptothecin (CPT, an apoptosis inducer) as positive control at the same time as OGD/R stimulation. TUNEL staining of L-02 cells under indicated conditions was performed (scale bar: 20 μm). (G-L) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 5 µM ferrostatin-1 (Fer-1, a ferroptotic inhibitor). L-02 cells were treated with 10 µM erastin (a ferroptotic inducer) as positive control at the same time as OGD/R stimulation. DCFH-DA staining was used to detect ROS production in cells (G). Fe2+ content in cells was measured by a commercial kit (H). The protein expression of GPX4 in cells was analyzed by immunofluorescence. Scale bar: 20 μm (I, K). Cell death was detected by propidium iodide (PI) staining. Scale bar: 50 μm (J, L). For statistical analysis, student’s t test and one-way ANOVA were used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Transfection, Over Expression, Plasmid Preparation, TUNEL Assay, Staining, Positive Control, Expressing, Immunofluorescence

Journal: World Journal of Diabetes

Article Title: Mechanism of the epidermal growth factor receptor in promoting endothelial cell dysfunction in gestational diabetes mellitus

doi: 10.4239/wjd.v16.i6.105173

Figure Lengend Snippet: High glucose promotes epidermal growth factor receptor expression by reducing ERRFI1 stability. A: Detection of the ubiquitination level of ERRFI1 ; B: ERRFI1 expression detected by Western blotting; C: ERRFI1 stability detected by cycloheximide chase; D: Binding between ERRFI1 and epidermal growth factor receptor (EGFR) detected by coimmunoprecipitation; E: Western blot showing ERRFI1 knockdown efficiency; F: Western blot showing EGFR expression. a P < 0.05. b P < 0.01. c P < 0.001. IP: Immunoprecipitation; IB: Immunoblotting; NC: Normal control; HG: High glucose; CHX: Cycloheximide; NC: Normal control; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; sh-NC: Short hairpin-negative control; sh- ERRFI1 : Short hairpin RNA targeting ERRFI1 ; EGFR: Epidermal growth factor receptor.

Article Snippet: The resulting lysate was subjected to immunoprecipitation with an anti- ERRFI1 antibody (1:300, Novus Biologicals) and was placed on a rotary shaker overnight at 4 °C.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Binding Assay, Knockdown, Immunoprecipitation, Control, Negative Control, shRNA

Journal: World Journal of Diabetes

Article Title: Mechanism of the epidermal growth factor receptor in promoting endothelial cell dysfunction in gestational diabetes mellitus

doi: 10.4239/wjd.v16.i6.105173

Figure Lengend Snippet: ERRFI1 binds to epidermal growth factor receptor and inhibits its activity to suppress high glucose-induced human umbilical vein endothelial cells dysfunction. A: Western blot showing ERRFI1 overexpression efficiency; B: Western blot showing epidermal growth factor receptor (EGFR) overexpression efficiency; C: Western blot showing EGFR expression in cells; D: Cell counting kit-8 assay showing cell viability; E: Cell apoptosis as detected by flow cytometry; F: Cell migration as assessed by scratch experiments; G: Cell migration as assessed by Transwell assay; H: Angiogenesis as assessed by tube formation assay; I: Western blot showing the expression of endothelial cell dysfunction markers. a P < 0.05. b P < 0.01. c P < 0.001. EGFR: Epidermal growth factor receptor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OE-NC: Overexpression-negative control; OE- ERRFI1 : Overexpression ERRFI1 ; NC: Normal control; OE-EGFR: Overexpression epidermal growth factor receptor; HG: High glucose; PI: Propidium iodide; FITC: Fluorescein isothiocyanate; VCAM-1: Vascular cell adhesion molecule-1; TNF-α: Tumor necrosis factor-α; VEGF-A: Vascular endothelial growth factor-A; ICAM-1: Intercellular cell adhesion molecule-1.

Article Snippet: The resulting lysate was subjected to immunoprecipitation with an anti- ERRFI1 antibody (1:300, Novus Biologicals) and was placed on a rotary shaker overnight at 4 °C.

Techniques: Activity Assay, Western Blot, Over Expression, Expressing, Cell Counting, Flow Cytometry, Migration, Transwell Assay, Tube Formation Assay, Negative Control, Control

Journal: Frontiers in Neuroscience

Article Title: Activation of the G Protein-Coupled Estrogen Receptor Elicits Store Calcium Release and Phosphorylation of the Mu-Opioid Receptors in the Human Neuroblastoma SH-SY5Y Cells

doi: 10.3389/fnins.2019.01351

Figure Lengend Snippet: E2 and G-1 stimulate phosphorylation of MOR in a PKC-dependent manner. (A,B) SH-SY5Y cells treated with PMA (1 μM), E2 (1 μM), or G-1 (1 μM) for 30 min showed increased level of phosphorylated MOR (pMOR) as compared with the vehicle group. In the presence of the pan-PKC inhibitor (Ro 31-8820, Ro, 3 μM), E2 and G-1 failed to increase pMOR expression. (C) The averaged pMOR level (relative to β-actin) in cells with different treatments. After detection of pMOR (A,B) , the nitrocellulose membrane was stripped and re-blocked by 5% fat-free dry milk in TBST, followed by incubation with MOR primary antibody, then MOR band was detected. ∗∗ P < 0.01, ∗∗∗ P < 0.001, one-way ANOVA with Tukey’s post hoc test, averaged data from three to four independent experiments.

Article Snippet: The membranes were blocked with 5% fat-free dry milk or 5% BSA (for detection of phosphorylated MOR, PKCα, Na + -K + -ATPase) in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h. Subsequently, the membranes were incubated with primary antibodies for 18 h at 4°C: rabbit GPER (1:1000, Abcam, Cat# ab39742, RRID:AB_1141090 ), rabbit anti-pMOR (1:1000, Cell Signaling Technology, Cat# 3451, RRID:AB_331619 ), rabbit anti-MOR (1:500, Novus, Cat# NBP1-31180, RRID:AB_2251717 ), rabbit anti-PKCα (1:1000, Cell Signaling Technology, Cat# 2056, RRID:AB_2284227 ), mouse anti-PKCε (1:1000, BD Biosciences, Cat# 610085, RRID:AB_397492 ), rabbit anti-Na + -K + -ATPase (1:3000, Abcam, Cat# ab76020, RRID:AB_1310695 ) and mouse anti-β-actin (1:2000, Bioworld Technology, BS6007M).

Techniques: Expressing, Incubation

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: MDA‐MB‐231 and SU86 cells were transfected with negative siRNA (siNeg) or indicated siRNA. Cell lysates were then blotted with the indicated antibodies. The mRNAs were extracted from the rest of the cells and subjected to qRT–PCR to determine knockdown efficiency. Error bars represent the SEM of three independent experiments. ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Cell lysates from 13 human cancer cell lines were blotted with anti‐EGFR antibody. Quantification of EGFR protein relative to the GAPDH was determined. Error bars represent the SEM of three independent experiments. MDA‐MB‐468, PANC1, MDA‐MB‐231, SU86, U251, and HCT116 cells were transfected with two ERRFI1 siRNAs; 48 h later, cell lysates were subjected to Western blot. The ratio of phospho‐AKT473 /total AKT signal (pAKT473/AKT) was quantified using ImageJ. pAKT473/AKT in siNeg is set to 1 within each cell line. Error bars represent ± SEM of three independent experiments. The significant difference between siNeg and siERRFI1 is indicated by: ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: MDA‐MB‐468 and U251 cell lysates were subjected to immunoprecipitation with control IgG or anti‐ERRFI1 antibody. The immunoprecipitates were blotted with the indicated antibodies. MDA‐MB‐468, PANC1, U251, and HCT116 cells were transfected with siERRFI1. Cell lysates were subjected to immunoprecipitation with control IgG or anti‐AKT antibody. The immunoprecipitates were blotted with the indicated antibodies. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Transfection

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: MDA‐MB‐468, PANC1, U251, and HCT116 cell lysates were subjected to immunoprecipitation with control IgG or anti‐ERRFI1 antibody. The immunoprecipitates were then blotted with the indicated antibodies. Quantifications of the Western blots were analyzed by ImageJ. To quantify each interaction, the amount of AKT or EGFR was first normalized back to the input level for each protein and then corrected by the amount of IPed protein, ERRFI1. Error bars represent ± SEM of three independent experiments. The significant difference between ERRFI1‐EGFR binding and ERRFI1‐AKT binding is indicated by: ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Purified recombinant GST, GST‐AKT, and His‐ERRFI1 were incubated in cell‐free conditions. The interaction between AKT and ERRFI1 was then examined. MDA‐MB‐468, PANC1, U251, and HCT116 cell lysates were subjected to immunoprecipitation with control IgG or anti‐PHLPP antibody. The immunoprecipitates were blotted with the indicated antibodies. The interaction was then quantified in each cell line. The amount of AKT corrected by IPed PHLPP was calculated in each of the four cancer cells transfected with siNeg or siERRFI1s. The results were then corrected by the siNeg. The interaction in siNeg is set to 1 within each cell line. Error bars represent ± SEM of three independent experiments. The significant difference between siNeg and siERRFI1 is indicated by: ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Purification, Recombinant, Incubation, Transfection

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: MDA‐MB‐468, PANC1, U251, and HCT116 cells were serum‐starved for 36 h and then treated with EGF (100 ng/ml) for 30 min prior to cell lysis. Cells lysates were subjected to immunoprecipitation with control IgG or anti‐ERRFI1 antibody. The immunoprecipitates were blotted with the indicated antibodies. The ERRFI1‐AKT and ERRFI1‐EGFR interaction was quantified using ImageJ. The immunoprecipitated ERRFI1 levels were normalized for each interaction and then corrected for MDA‐MB‐468 no EGF treatment. The interaction in no EGF‐treated (‐EGF) MDA‐MB‐468 is set to 1. Error bars represent ± SEM of three independent experiments. The significant difference between vehicle and EGF treatment is indicated by: ** P < 0.01. Statistical analyses were performed with Student's t ‐test. U251 and HCT116 cells were transfected with ERRFI1 construct. After 48 h, cells were lysed and subjected to immunoprecipitation with control IgG or anti‐PHLPP antibody. The immunoprecipitates were then blotted with the indicated antibodies. U251 and HCT116 cells from (B) were treated with increasing dose of gemcitabine (Gem) alone or in combination with 10 μM MK‐2206 2HCl (MK) for 72 h. Cell survival was then determined. Each point shows the mean values for three independent experiments; error bars represent ± SEM. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Lysis, Immunoprecipitation, Transfection, Construct

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: MDA‐MB‐468, PANC1, U251, and HCT116 cells lysates were subjected to immunoprecipitation with control IgG, anti‐AKT1, anti‐AKT2, or anti‐AKT3 antibody. The immunoprecipitates were blotted with the indicated antibodies. The supernatant of the precipitation were blotted with AKT1, AKT2, and AKT3 antibodies to ensure that the majority of the AKT isoform was precipitated with specific antibody. Quantification analysis of all three AKT isoforms. In the input sample, each AKT isoform was normalized to GAPDH, and then the normalized levels of AKT2 and AKT3 relative to AKT1 were determined within each cell line. Error bars represent ± SEM of three independent experiments. Quantifications of the interaction between ERRFI1 and three AKT isoforms using ImageJ. Antibodies against three AKT isoforms was used to pull down ERRFI1. The ERRFI1‐AKT isoform interactions were determined in a similar fashion as Fig C. Error bars represent ± SEM of three independent experiments. The significant difference between AKT2/or AKT3‐ERRFI1 and AKT1‐ERRFI1 interaction is indicated by: ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Cells lysates from (A) were subjected to immunoprecipitation with control IgG or anti‐ERRFI1 antibody. The immunoprecipitates were blotted with the indicated antibodies. The interactions were quantified in a similar fashion as in (C). Error bars represent ± SEM of three independent experiments. The significant difference between AKT2/or AKT3‐ERRFI1 and AKT1‐ERRFI1 interaction is indicated by: ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Immunoprecipitation

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: A–D MDA‐MB‐468 (A), PANC1 (B), U251 (C), and HCT116 (D) were transfected with siNeg or siERRFI1. Cell lysates were subjected to immunoprecipitation with control IgG or anti‐PHLPP1 antibody. The immunoprecipitates were blotted with the indicated antibodies. AKT‐PHLPP interaction was quantified as described before. Error bars represent ± SEM of three independent experiments. The significant difference between siNeg and siERRFI1 is indicated by: * P < 0.05, ** P < 0.01. Statistical analyses were performed with Student's t ‐test. E U251 cells were transfected with GST‐tagged full‐length ERRFI1 (FL) or ERRFI1 truncation constructs; 48 h later, half of the cells were harvested and lysates were subjected to immunoprecipitation with control IgG or anti‐GST antibody. The immunoprecipitates were then blotted with the indicated antibodies. F The rest of the U251 cells from (E) were treated with increasing doses of TCN or gemcitabine for 3 days. Survival fraction was determined by CYQUANT assay. The x ‐axis indicates drug dose, and the y ‐axis indicates the survival fraction after drug exposure. Overexpression efficiency is shown in (E, input). Error bars represent ± SEM of three independent experiments. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Transfection, Immunoprecipitation, Construct, CyQUANT Assay, Over Expression

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: A, B Knockdown of ERRFI1 increased proliferation and colony formation in MDA‐MB‐468 and PANC1, two EGFR‐high cells (A). Knockdown of ERRFI1 decreased proliferation and colony formation in U251 and HCT116 EGFR‐low cells (B). Cell proliferation was monitored every 12 h. The x ‐axis indicates time post‐treatment, and the y ‐axis indicates cell proliferation rate. The representative colony formation pictures from triplicate experiments are shown. Error bars represent ± SEM of three independent experiments; * P < 0.05, ** P < 0.01 compared to siNeg. Statistical analyses were performed with Student's t ‐test. Remaining cells were blotted with the indicated antibodies. C, D U251 and HCT116 cells were transfected with indicated siRNA. Cell lysates were blotted with the indicated antibodies. Cell proliferation and colony formation were assessed. Quantification of colony formation is shown. Error bars represent ± SEM of three independent experiments. ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Transfection

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: A MDA‐MB‐468, PANC1, U251, and HCT116 cells transfected with siERRFI1 were treated with increasing doses of gemcitabine for 72 h, and cell survival was then determined. The x ‐axis indicates drug dose, and the y ‐axis indicates the survival fraction after gemcitabine exposure. Knockdown efficiency is shown in (D). Error bars represent the SEM of three independent experiments. Statistical analyses were performed with Student's t ‐test. B MDA‐MB‐468 and PANC1 cells transfected with siERRFI1 were treated with increasing doses of gemcitabine (Gem) alone or in combination with either 10 μM gefitinib (G) or 10 μM TCN (T) for 72 h. Cell survival was then determined. Knockdown efficiency is shown in (D). Error bars represent the SEM of three independent experiments. Statistical analyses were performed with Student's t ‐test. C U251 and HCT116 transfected with ERRFI1 plasmid were treated with Gem alone or in combination with either 10 μM T or 10 μM G for 72 h. Cell survival was then determined. Overexpression efficiency is shown in (E). Error bars represent the SEM of three independent experiments. Statistical analyses were performed with Student's t ‐test. D, E MDA‐MB‐468 and U251 cells from (B and C) were treated with vehicle or 20 nM of Gem alone or in combination with either 10 μM of G or 10 μM of T for 12 h. Cells lysates were blotted with the indicated antibodies. The pAKT473/AKT ratio and pERK473/ERK ratio were quantified. The ratio in siNeg or EV is set to 1 within each cell line. Data represent the SEM of three independent experiments. ** P < 0.01. Statistical analyses were performed with Student's t ‐test. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Over Expression

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: A Organoid lysates from six human breast cancer PDX tumors were blotted with EGFR antibody. Quantification of EGFR protein relative to the GAPDH was determined. Error bars represent the SEM of three independent experiments. B, C EGFR‐high BJ06 and BJ16 organoids transfected with siERRFI1 were treated with vehicle (Veh) or 50 nM of gemcitabine (Gem) alone or in combination with either 10 μM gefitinib (G) or 10 μM TCN (T). Organoid growth was monitored every 3 days. Knockdown efficiency is shown in (F). Error bars represent the SEM of three independent experiments. ** P < 0.01. Statistical analyses were performed with Student's t ‐test. D, E EGFR‐low BJ43 and BJ44 organoids transfected with ERRFI1 plasmid were treated as above. Organoid growth was monitored every 3 days. Overexpression efficiency is shown in (G). Error bars represent the SEM of three independent experiments. ** P < 0.01. Statistical analyses were performed with Student's t ‐test. F, G BJ06 and BJ44 organoids were treated with vehicle or 50 nM of Gem alone or in combination with either 10 μM of G or 10 μM of T for 72 h. Organoid lysates were blotted with the indicated antibodies. Source data are available online for this figure.

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Over Expression

Journal: EMBO Reports

Article Title: Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

doi: 10.15252/embr.201744767

Figure Lengend Snippet: MTS assay of candidate genes selected for siRNA screening

Article Snippet: A second siRNA for ERRFI1 was purchased from OriGene (Rockville, MD).

Techniques: MTS Assay, Selection

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: Primers used for SYBR green-based quantitative real-time PCR analysis for gene expression

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: Real-time Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: Primers used for generating pGL4.23- ERRFI1 downstream enhancer reporter and pLKO.1-sh ERRFI1 construct

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques:

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: ERRFI1 is downregulated in breast cancer tumor samples and is differentially induced by CORT in TNBC. (A) Genotype-Tissue Expression Project (GTEx) data for normal breast tissue and the Cancer Genome Atlas breast invasive carcinoma RNA-seq data (from the Cancer Genome Atlas Research Network: http://www.cancer.gov/tcga ) were analyzed using the online tool Gene Expression Profiling Interactive Analysis . ERRFI1 is significantly downregulated in breast cancer samples compared to normal tissue equivalents ( P < 0.01). (B) MCF10A, MDA-MB-468, and MDA-MB-231 cells (n= 3-4/treatment) were treated with increasing doses of CORT for 2 h. In MCF10A cells, dose-dependent increase in ERRFI1 mRNA expression levels was observed upon CORT treatment (1-way ANOVA; F(5,17) = 573.4; P < 0.0001). In MDA-MB-468 cells, a significant increase in ERRFI1 mRNA was observed starting at 100 nM CORT and did not change with increasing CORT dose (1-way ANOVA; F(5,17) = 11.55; P < 0.0001). In MDA-MB-231, ERRFI1 mRNA levels increased with increasing CORT dose (1-way ANOVA; F(5,18) = 63.67; P < 0.0001) but with a lower magnitude of induction compared to MCF10A cells. (C) All 3 TNBC lines were treated with 100 nM CORT for the timepoints indicated before harvest and real-time quantitative PCR analysis. In MCF10A and MDA-MB-468, a significant increase in ERRFI1 expression level relative to the vehicle-treated control was observed after 1 h (Student’s t -test; MCF10A, P < 0.0001; MDA-MB-468, P = 0.01) while in MDA-MB-231, ERRFI1 mRNA expression significantly increased after 30 mins (Student’s t -test; MDA-MB-231: P = 0.0179). (D) All 3 TNBC lines expressed similar levels of NR3C1 ( GR ) mRNA (1-way ANOVA; F(2,8) = 0.9548; P = 0.4248). ERRFI1 mRNA levels were normalized to the reference gene 18s rRNA , which was unaffected by hormone treatment. The normalized values were log 10 transformed prior to statistical analysis. Dose-response curves were fitted by nonlinear regression. Dots represent the log 2 (fold induction) ± standard error of the mean while bars represent mean ± standard error of the mean with statistical significance indicated by asterisks in Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001) or letters above the means (lowercase for MCF10A, uppercase for MDA-MB-231, and lowercase with an overline for MDA-MB-468) in 1-way ANOVA (means with the same letter are not significantly different; Tukey’s multiple comparison test; P < 0.05). Experiments were performed twice with consistent results and graphs shown are representative of the different trials.

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: Expressing, RNA Sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Control, Transformation Assay, Comparison

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: Induction of ERRFI1 mRNA is resistant to protein synthesis inhibition and is abolished in the presence of a selective-GR antagonist. (A) MCF10A cells were incubated with 100 µg/mL CHX for 30 min before addition of CORT (300 nM). Treatment with CORT plus CHX was continued for 2 h before cell harvest for analysis of ERRFI1 mRNA. Treatment with CORT significantly induced ERRFI1 mRNA expression and CORT-dependent induction persisted in the presence of CHX (Student’s t -test; −CHX, P = 0.0001; +CHX, P < 0.0001). (B) CORT treatment of MDA-MB-231 cells significantly increased ERRFI1 mRNA expression and is resistant to protein synthesis inhibition (Student’s t -test; −CHX, P < 0.0001; +CHX, P = 0.0006). CORT treatment likewise caused a statistically significant increase in ERRFI1 pre-mRNA levels in (C) MCF10A (Student’s t -test; −CHX, P < 0.0001; +CHX, P < 0.0001) and in (D) MDA-MB-231 cells (Student’s t -test; −CHX, P < 0.0001; +CHX, P = 0.0099) that persisted with CHX treatment. Pre-incubation with 1 µM of the GR-selective antagonist mifepristone (MIF; RU486) for 1 h before addition of vehicle or CORT (100 nM) for 2 h abolished the CORT-dependent induction of ERRFI1 mRNA in (E) MCF10A (1-way ANOVA; F(2,9) = 268.8, P < 0.0001) and (F) MDA-MB-231 (1-way ANOVA; F(2,9) = 78.92, P < 0.0001). ERRFI1 mRNA levels were normalized to the 18s rRNA housekeeping gene whose expression was not affected by hormone treatment, and normalized values were log 10 transformed before statistical analysis. Bars represent the fold induction ± standard error of the mean relative to vehicle control with statistical significance indicated by asterisks in Student’s t -test (* P < 0.01, ** P < 0.001, *** P < 0.0001) or letters above the means in 1-way ANOVA (means with the same letter are not significantly different; Tukey’s multiple comparison test; P < 0.05). All treatments were done with 3 to 4 replicates. Experiments were performed at least twice with consistent results. Graphs shown are representative of the different trials.

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: Inhibition, Incubation, Expressing, Transformation Assay, Control, Comparison

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: Identification of an 821-bp enhancer element downstream of the ERRFI1 TSS that supports GR-mediated transactivation. (A) The UCSC Genome Browser with annotation based on the human February 2009 (GRCh37/hg19) genome assembly was used to visualize the human ERRFI1 locus and surrounding nongenomic regions. Highlighted are the candidate ERRFI1 downstream enhancer (EDE) region located ~21.5 kb downstream of the ERRFI1 TSS and the proximal promoter region. (B) The EDE contains 2 GREs predicted by LASAGNA 2.0 . MDA-MB-231 GR- (GSE56022) , and MCF10A GR- (GSE102355) and RNA Pol II ChIP-seq data (GSE94062) were obtained from the Gene Expression Omnibus. The putative enhancer is a transcription factor binding hotspot, which is suggestive of a regulatory function and is enriched for active enhancer marks as demonstrated by analysis of ENCODE data for noncoding transcripts based on RNA-seq, H3K27Ac ChIP-seq peaks, DNase I hypersensitivity clusters, vertebrate conservation, and predicted long-range interactions from the GeneHancer database . Enhancer luciferase constructs containing the 821-bp EDE or empty vector control were transfected into (C) MCF10A and (D) MDA-MB-468 cells. Twenty hours after transfection, cells were treated with vehicle (100% ethanol), CORT (300 nM), or RU486 (1 uM) plus CORT for 20 h before harvest and analysis by dual luciferase assay. In MCF10A and MDA-MB-468 cells, the EDE showed robust CORT-dependent transactivation that was abolished with RU486 treatment (1-way ANOVA; MCF10A: F(2,9) = 180.1, P < 0.0001; MDA-MB-468: F(2,9) = 24.72, P = 0.0002). The empty vector control showed slight CORT-induced transactivation that was not affected by RU486 (1-way ANOVA; MCF10A: F(2,9) = 6.075, P = 0.0214; MDA-MB-468: F(2,7) = 4.560, P = 0.0540). Relative luminescence units were obtained from Firefly luminescence normalized to the Renilla control whose activity did not change across treatments. Normalized values were log 10 transformed before statistical analysis. Bars represent mean ± standard error of the mean and the letters above the mean indicate significant differences among treatments (means with the same letter are not significantly different; P < 0.05, Tukey’s multiple comparison test). All treatments were done with 3 to 4 biological replicates and all experiments were performed twice with consistent results.

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: ChIP-sequencing, Gene Expression, Binding Assay, RNA Sequencing, Luciferase, Construct, Plasmid Preparation, Control, Transfection, Activity Assay, Transformation Assay, Comparison

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: ERRFI1 knockdown has different effects on cell survival, proliferation, and viability in TNBC. Effects of ERRFI1 knockdown and CORT treatment on (A-C) cell survival, (D-F) direct cell proliferation, and (G-I) cell viability of TNBC cells were evaluated using assays based on colony formation, fluorescence-based DNA-binding, and resazurin reduction, respectively. For the colony formation assay, scrambled and sh ERRFI1 cells were treated with vehicle (100% ethanol) or CORT (100 nM) for 14 days. For the direct cell proliferation and cell viability assay, cells were treated as previously described for 72 h. In MCF10A, CORT (A) enhanced colony formation (2-way ANOVA; Treatment factor: F(1,8) = 201.0, P < 0.0001; Knockdown factor: F(1,8) = 189.4, P < 0.0001), (D) increased cell proliferation (2-way ANOVA; Treatment factor: F(1,15) = 110.2, P < 0.0001; Knockdown factor: F(1,15) = 239.3, P < 0.0001), and (G) increased cell viability (2-way ANOVA; Treatment factor: F(1,14) = 97.85, P < 0.0001; Knockdown factor: F(1,14) = 118.2, P < 0.0001). These effects of CORT were enhanced by ERRFI1 knockdown. CORT treatment of MDA-MB-468 cells (B) did not affect cell survival (2-way ANOVA; Treatment factor: F(1,8) = 3.615, P = 0.0938; Knockdown factor, F(1,8) = 62.04, P < 0.0001), (E) slightly augmented cell proliferation (2-way ANOVA; Treatment factor: F(1,12) = 13.38, P = 0.0033; Knockdown factor: F(1,12) = 6.215, P = 0.0283), (H) and increased cell viability (2-way ANOVA; Treatment factor: F(1,14) = 158.3, P < 0.0001; Knockdown factor: F(1,14) = 28.99, P < 0.0001). Knockdown of ERRFI1 increased cell survival and viability independent of the pro-tumorigenic effects of CORT but had no effect on cell proliferation. In MDA-MB-231, CORT treatment (C) promoted colony formation (2-way ANOVA; Treatment factor: F(1,8) = 71.64, P < 0.0001; Knockdown factor: F(1,8) = 54.05, P < 0.0001), (F) enhanced proliferation (2-way ANOVA; Treatment factor: F(1,13) = 13.56, P = 0.0028; Knockdown factor: F(1,13) = 16.52, P = 0.0013), and (I) increased viability (2-way ANOVA; Treatment factor: F(1,16) = 31.27, P < 0.0001; Knockdown factor: F(1,16) = 61.37, P < 0.0001). Knockdown of ERRFI1 conferred anti-tumorigenic effect in the cell line. For the cell proliferation and cell viability assays, measures were normalized to raw fluorescence reads at 0 h, which was set as baseline (relative fluorescence units), and normalized values were log 10 transformed before statistical analysis. Bars represent mean ± standard error of the mean with statistical significance determined through Student’s t- test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 for statistically significant effects of CORT within a shRNA type, and # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 for statistically significant effects of ERRFI1 knockdown between the same hormone treatment). For the colony formation assay, treatments were performed with 3 replicates, while for the direct cell proliferation and cell viability assays, treatments were done with 5 replicates. All experiments were performed at least twice with consistent results and graphs shown are representative of the different trials.

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: Knockdown, Fluorescence, Binding Assay, Colony Assay, Viability Assay, Transformation Assay, shRNA

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: ERRFI1 knockdown has different effects on cell migration and apoptosis of TNBC. Assessment of cell migration (A and B) was performed using the wound healing assay. Cells were treated with either vehicle (100% ethanol) or CORT (500 nM). Representative images of the scratch at each timepoint are shown to the right of each plot. (A) In MDA-MB-468, CORT treatment of scrambled shRNA-transduced cells increased migration but had no effect on sh ERRFI1 -transduced cells. Knockdown of ERRFI1 , however, increased cell migration compared to scrambled shRNA control cells with the same treatment (2-way ANOVA; Treatment factor: F(1,14) = 12.05, P = 0.0037; Knockdown factor: F(1,14) = 111.0, P < 0.0001). (B) In MDA-MB-231 cells, CORT treatment-enhanced cell migration of scrambled shRNA- and sh ERRFI1 -transduced cells. However, ERRFI1 knockdown decreased cell migration compared to scrambled shRNA control cells with the same hormone treatment (2-way ANOVA; Treatment factor: F(1,14) = 50.83, P < 0.0001; Knockdown factor: F(1,14) = 48.83, P < 0.0001). Apoptosis (C and D) of scrambled and ERRFI1 knockdown cells was assessed using an assay based on the activity of caspase-3 and caspase-7 to cleave an amino acid peptide conjugated to a DNA-binding dye. Cells were treated with vehicle, CORT (100 nM), DOX (500 nM in MDA-MB-468; 5 µM in MDA-MB-231) and a combination of CORT plus DOX (n = 4/treatment) and apoptosis was measured 48 h after treatment. (C) In MDA-MB-468 cells, CORT enhanced DOX-induced apoptosis of scrambled shRNA-transduced cells. Upon ERRFI1 knockdown, CORT had a protective effect against DOX-induced apoptosis (2-way ANOVA; Treatment factor: F(3,21) = 211.5, P < 0.0001; Knockdown factor: F(1,21) = 4.762, P = 0.0406). (D) In MDA-MB-231 cells, ERRFI1 knockdown sensitized cells to the apoptotic effect of DOX, while CORT treatment suppressed the DOX-induced cytotoxicity (2-way ANOVA; Treatment factor: F(3,23) = 23.10, P < 0.0001; Knockdown factor: F(1,23) = 96.45, P < 0.0001). For the wound healing assay, percentage wound closure was designated as the average of the area difference of the 2 images taken in each well relative to the baseline measure at 0 h. These values were log 10 transformed before statistical analysis. Bars represent mean ± standard error of the mean with statistical significance determined through Student’s t- test (* P < 0.01,** P < 0.001 for statistically significant effects of treatment within a shRNA type, and # P < 0.01, ## P < 0.001, ### P < 0.0001 for statistically significant effects of ERRFI1 knockdown between the same treatment) or through 2-way ANOVA followed by Tukey’s multiple comparisons test (bars with same letters above the means are not significantly different; P < 0.05). Treatments were performed with 4 to 5 replicates for both assays. All experiments were performed at least twice with consistent results and graphs shown are representative of the different trials.

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: Knockdown, Migration, Wound Healing Assay, shRNA, Control, Activity Assay, Binding Assay, Transformation Assay

Journal: Endocrinology

Article Title: Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

doi: 10.1210/endocr/bqaa082

Figure Lengend Snippet: Overexpression of m Errfi1 differentially alters cell survival and viability of TNBC. Effects of CORT treatment and m Errfi1 overexpression on cell survival, and cell viability of MDA-MB-468 and MDA-MB-231 cells. Clonogenic assay (A and C) on empty or m Errfi1 overexpressing cells (n = 3/treatment) treated with either vehicle or CORT (100 nM) for 14 days. Representative images of colonies stained with crystal violet are shown to the right of each plot. Cell viability (n = 4-5/treatment) (B and D) was assessed using resazurin-based assay. Cells transduced with empty or m Errfi1 -overexexpression vector (n = 4-5/treatment) were treated with vehicle or CORT (100 nM) for 72 h. In MDA-MB-468 cells, CORT treatment increased (A) cell survival (2-way ANOVA; Treatment factor: F(1,8) = 32.11, P < 0.0005; Overexpression factor: F(1,8) = 186.8, P < 0.0001) and (B) viability (2-way ANOVA; Treatment factor: F(1,8) = 16.53, P = 0.0012; Overexpression factor: F(1,8) = 38.51, P < 0.0001). Overexpression of m Errfi1 led to marked decrease in cell survival and viability. In MDA-MB-231 cells, CORT treatment significantly enhanced (C) colony formation (2-way ANOVA; Treatment factor: F(1,8) = 312.4, P < 0.0001; Overexpression factor: F(1,8) = 1.081, P = 0.3289) and (D) cell viability (2-way ANOVA; Treatment factor: F(1,16) = 14.53, P = 0.0015; Overexpression factor: F(1,16) = 0.1916, P = 0.6675). Overexpression of m Errfi1 did not alter these cellular behaviors. For cell viability assay, measures were normalized to raw fluorescence reads at 0 h, which was set as baseline (relative fluorescence units). Normalized values were log 10 transformed before statistical analysis. Bars represent mean ± standard error of the mean with statistical significance determined through Student’s t- test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 for statistically significant effects of CORT within one expression vector, and # P < 0.01, ## P < 0.001 for statistically significant effects of ERRFI1 overexpression between the same hormone treatment). Experiments were performed at least twice with consistent results and graphs shown are representative of the different trials.

Article Snippet: The short hairpin RNA (shRNA) construct targeting ERRFI1 (sh ERRFI1 ; TRCN0000118131; Genetic Perturbation Platform shRNA library, Broad Institute) and scrambled shRNA pLKO.1 (RRID:Addgene_1864) ( ) were used for lentiviral production.

Techniques: Over Expression, Clonogenic Assay, Staining, Resazurin Assay, Transduction, Plasmid Preparation, Viability Assay, Fluorescence, Transformation Assay, Expressing