Journal: Molecular Cancer Therapeutics

Article Title: MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling

doi: 10.1158/1535-7163.MCT-23-0218

Figure Lengend Snippet: Phosphorylation of MIG6 Y394 and Y395 is decreased in ALK/ROS1 fusion-driven cells by crizotinib treatment. A, Schematic workflow of phosphoproteomics for H3122 and CUTO28 cells under 2-hour crizotinib treatment to explore the early adaptive signaling reprogramming under ALK/ROS1 inhibition. B, Proteins with phosphorylation sites significantly regulated by crizotinib (|FC| > 1.5, P < 0.05) in either phosphotyrosine (pY) enriched phosphoproteomics or global phosphoproteomics (pSTY) were integrated and analyzed by GO enrichment analysis. The Top 10 GO enrichments in crizotinib-regulated phosphoproteins were displayed. 27 phosphoproteins (displayed in their corresponding gene names) with significant phosphorylation changes under crizotinib treatment in CUTO28 cells, were enriched in the GO term “regulation of ERBB signaling pathway”. C, Protein–protein interaction network analysis for the 27 genes identified in B to explore potential EGFR regulators. Very strong interactions (STRING database protein-protein interaction confidence score > 0.9) were highlighted in thick edges. Residues with significantly increased or decreased phosphorylation are indicated with dark red or dark blue, respectively. D and E, Volcano plots showing decreased phosphorylation of MIG6 Y394 and Y395 under crizotinib treatment in H3122 and CUTO28 pY-enriched phosphoproteomics. Significantly regulated phosphoproteins (|FC| > 1.5, P < 0.05) were colored in red. F and G, Immunoblots showing sustained MIG6 protein levels in H3122 and CUTO28 treated with 250 nmol/L or 100 nmol/L crizotinib for 2 hours, respectively.

Article Snippet: WT MIG6 lentiviral plasmid (#RC206883L3) and the empty vector (#PS100092) were purchased from Origene.

Techniques: Phospho-proteomics, Inhibition, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling

doi: 10.1158/1535-7163.MCT-23-0218

Figure Lengend Snippet: MIG6 protein levels are decreased following extended ALK/ROS1 inhibition and upfront inhibition of ERBB signaling enhances responses to ALK/ROS1 inhibitors. H3122 cells ( A ) were treated with 250 nmol/L crizotinib or 50 nmol/L alectinib, and CUTO28 cells ( B ) were treated with 250 nmol/L crizotinib or 100 nmol/L entrectinib for indicated time points. Lysates were immunoblotted with indicated antibodies. C and D, H3122 and CUTO28 were treated with various ALK and ROS1 inhibitors alone or in combination with 100 nmol/L afatinib for 72 hours and then assayed with MTS for cell viability. Data represent the mean ± SEM for three biological replicates. E and F, H3122 and CUTO28 cells were treated with DMSO, ALK or ROS1 inhibitors (500 nmol/L crizotinib or 100 nmol/L alectinib for H3122; 100 nmol/L crizotinib or 100 nmol/L entrectinib for CUTO28) alone or in combination with 100 nmol/L afatinib for 6 hours. Cell lysates were immunoblotted with indicated antibodies. pERK with high-intensity exposure was shown as pERK (high). G and H, H3122 and CUTO28 cells were seeded 2,000 cells/well in a 12-well plate and treated with DMSO, ALK/ROS1 inhibitors (125 nmol/L crizotinib or 25 nmol/L alectinib for H3122, 25 nmol/L crizotinib or 12.5 nmol/L entrectinib for CUTO28) alone or in combination with 100 nmol/L afatinib for two weeks. Colonies were stained by crystal violet.

Article Snippet: WT MIG6 lentiviral plasmid (#RC206883L3) and the empty vector (#PS100092) were purchased from Origene.

Techniques: Inhibition, Staining

Journal: Molecular Cancer Therapeutics

Article Title: MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling

doi: 10.1158/1535-7163.MCT-23-0218

Figure Lengend Snippet: MIG6 knockdown induces resistance to ALK/ROS1 inhibition via the EGFR–SHC1–MAPK pathway. A and B, H3122 and CUTO28 were transduced with two different shRNA targeting MIG6 or nontargeting control (NTC) shRNA. Cells were treated with ALK/ROS1 inhibitors alone or in combination with 100 nmol/L afatinib for 72 hours, and then assayed by MTS for cell viability. Data represent the mean ± SEM for three biological replicates. C and D, As in A and B , except that cells were treated with afatinib alone . E and F, H3122 and CUTO28 NTC and MIG6 knockdown cells were treated with crizotinib alone (500 nmol/L for H3122 and 100 nmol/L for CUTO28) or in combination with 100 nmol/L afatinib for 2 hours. Lysates were immunoblotted with indicated antibodies. G, H3122 NTC and MIG6 knockdown cells were stimulated with 10 ng/mL EGF for 15 minutes. Cell lysates were subjected to immunoprecipitation (IP) using EGFR antibody and were immunoblotted for SHC1. In parallel, whole cell lysates (WCL) were immunoblotted with indicated antibodies.

Article Snippet: WT MIG6 lentiviral plasmid (#RC206883L3) and the empty vector (#PS100092) were purchased from Origene.

Techniques: Knockdown, Inhibition, Transduction, shRNA, Control, Immunoprecipitation

Journal: Molecular Cancer Therapeutics

Article Title: MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling

doi: 10.1158/1535-7163.MCT-23-0218

Figure Lengend Snippet: MIG6 downregulation is sustained in cells with EGFR-mediated acquired resistance to ALK/ROS1 inhibition. A, H3122 and crizotinib-resistant (CR1-CR3) cell lysates were immunoblotted with indicated antibodies. B and C, H3122 and CR1-CR3 cells were treated with crizotinib alone or in combination with 100 nmol/L afatinib for 72 hours and assayed with MTS for cell viability. Data represent the mean ± SEM for three biological replicates. D, Parental and resistant H3122 cells were treated with DMSO, 250 nmol/L crizotinib, 100 nmol/L afatinib alone, or a combination of crizotinib and afatinib for 2 hours. Lysates were immunoblotted with indicated antibodies. E and F, CUTO37 and entrectinib-resistant CUTO37-ER cells were treated with entrectinib or afatinib alone for 72 hours and assayed with MTS for cell viability. Data represent the mean ± SEM for three biological replicates. G, CUTO37 and ER cells were treated with DMSO, 100 nmol/L entrectinib, 100 nmol/L afatinib alone, or a combination of crizotinib and afatinib for 2 hours. Lysates were immunoblotted with indicated antibodies. All the resistant cells were seeded without TKIs for 24 hours prior to drug treatments for immunoblots.

Article Snippet: WT MIG6 lentiviral plasmid (#RC206883L3) and the empty vector (#PS100092) were purchased from Origene.

Techniques: Inhibition, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling

doi: 10.1158/1535-7163.MCT-23-0218

Figure Lengend Snippet: MIG6 expression and phosphorylation are critical for suppressing EGFR-mediated acquired resistance to ALK/ROS1 inhibition. A, H3122 CR1 transduced with empty vector (EV), wild-type (WT) MIG6, or Y394F/Y395F mutant MIG6 lentivirus were treated with crizotinib for 72 hours and assayed with MTS for cell viability. Data represent the mean ± SEM for three biological replicates. Two-way ANOVA was performed to show the statistically significant difference of crizotinib responsiveness between WT MIG6 and mutant MIG6–expressing H3122 CR1 cells (***, P < 0.001). B, Cells were treated with DMSO or 250 nmol/L crizotinib for 2 hours and lysates were immunoblotted with indicated antibodies. Overexpressed Myc-tagged MIG6 was detected by the MIG6 antibody. C, CUTO37 ER cells were transduced with empty vector (EV), wild-type (WT) MIG6, or Y394F/Y395F mutant MIG6 lentivirus. After one week of puromycin selection, cells were cultured for another week free of entrectinib incubation. Colonies were stained with crystal violet and quantified in D . Data represent the mean ± SEM for three biological replicates. One-way ANOVA with Tukey post hoc test was performed to calculate P values (*, P < 0.05; **, P < 0.01; ns, no significance). E, CUTO37 ER cells transduced with empty vector (EV), WT MIG6 or Y394F/Y395F mutant MIG6 lentivirus were treated with DMSO or 100 nmol/L entrectinib for 2 hours. Lysates were immunoblotted with indicated antibodies.

Article Snippet: WT MIG6 lentiviral plasmid (#RC206883L3) and the empty vector (#PS100092) were purchased from Origene.

Techniques: Expressing, Phospho-proteomics, Inhibition, Transduction, Plasmid Preparation, Mutagenesis, Selection, Cell Culture, Incubation, Staining

Journal: Molecular Cancer Therapeutics

Article Title: MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling

doi: 10.1158/1535-7163.MCT-23-0218

Figure Lengend Snippet: High EGFR to MIG6 ratios and MIG6-damaging alterations correlate with resistance to ALK/ROS1 inhibition. A, Scatter plot showing a positive correlation between EGFR to MIG6 ratios and crizotinib IC 50 s in ALK/ROS1 cell lines (Pearson correlation coefficient R = 0.81, P = 0.0023). Fragments Per Kilobase Million (FPKM) normalized counts of EGFR and MIG6 (encoded by ERRFI1 ) from the RNA-seq data of 11 ALK/ROS1 lines were used to calculate the EGFR to MIG6 expression ratios. These cell lines include 7 ROS1 lines (patient-derived CUTO23, 27, 28, 33, 37, and 38 lines, and HCC78), 1 ALK line (H3122), 1 ALK-resistant line (H3122-CR1), and 2 ROS1-resistant lines (HCC78-TR and CUTO37-ER). Crizotinib IC 50 s were derived from 3-day MTS assays. B, Violin plot of EGFR to MIG6 ratios in ALK inhibitor (ALKi) naïve and unpaired ALKi-resistant tumors from the Wilson cohort. EGFR to MIG6 ratios were calculated using Reads Per Kilobase Million (RPKM) values from the RNA-seq data. The P value was determined by unpaired Student t test. C, Violin plot of EGFR to MIG6 ratios in pre-ALKi and unpaired post-ALKi–treated tumors from the Caris cohort. EGFR to MIG6 ratios were calculated using transcripts per million (TPM) values from the RNA-seq data. Due to the unequal variance between the two groups ( F test with P < 0.0001), unpaired Welch's t test was performed to determine the P value. D, Schematic diagram of MIG6 truncation mutation 241Q* in CUTO63 cells harboring SLC34A2-ROS1 (S13:R32, S13:R34). This mutation results in a complete deletion of the EGFR-binding domain (residues 337–412) of MIG6. E, MIG6 immunoblotting analysis in a panel of ROS1 parental and resistant cells. Full-length MIG6 was probed by an antibody raised against MIG6 residues 156–280 (Santa Cruz Biotechnology) while the truncated MIG6 was probed by an antibody raised against MIG6 residues 111–221 (Abnova) before the truncation mutation site. High-intensity exposure was used to detect the low-level MIG6 in CUTO37-ER cells. F, CUTO63 cells were treated with indicated inhibitors for 72 hours and assayed with MTS. Data represent the mean ± SEM for three biological replicates. G, CUTO63 cells were treated with a dose range of afatinib or entrectinib for 2 hours. Lysates were immunoblotted with indicated antibodies. H, CUTO63 cells were transduced with doxycycline-inducible wild-type (WT) MIG6 or Y394F/Y395F–mutant MIG6 lentivirus. CUTO63 cells were treated with or without 100 ng/mL doxycycline for 24 hours and then lysates were harvested and immunoblotted with indicated antibodies. I, Timeline of treatments and biopsies for an NSCLC patient (#MSK-P-0012008) harboring an SDC4-ROS1 fusion. ROS1 inhibitors are highlighted in blue. J, Schematic of the intragenic MIG6 deletion detected in the second biopsy of the patient in I after ROS1 inhibitor treatments, showing the MIG6 exons 3 and 4, including the EGFR-binding domain, were completely deleted. The other allele of ERRFI1 was lost, suggesting a loss of heterozygosity (LOH) at this gene.

Article Snippet: WT MIG6 lentiviral plasmid (#RC206883L3) and the empty vector (#PS100092) were purchased from Origene.

Techniques: Inhibition, RNA Sequencing, Expressing, Derivative Assay, Mutagenesis, Binding Assay, Western Blot, Transduction

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: 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: Biology of Reproduction

Article Title: ERBB Receptor Feedback Inhibitor 1 Regulation of Estrogen Receptor Activity Is Critical for Uterine Implantation in Mice 1

doi: 10.1095/biolreprod.109.081307

Figure Lengend Snippet: The expression of Errfi1 by real-time RT-PCR and in situ hybridization in pseudopregnancy. A) The expression level of Errfi1 was measured in uteri of pseudopregnancy. Total RNA used for the RT-PCR assays was prepared from pseudopregnant uteri. The results represent the mean ± SEM of three independent RNA sets. **P < 0.01 and ***P < 0.001. B) The localization pattern of Errfi1 mRNA by in situ hybridization during early pregnancy. Nuclei are counterstained with hematoxylin. E, embryonic day; EM, embryo; LE, luminal epithelium; GE, glandular epithelium; PDZ, primary decidual zone; SDZ, secondary decidual zone; ST, stroma cells; MYO, myometrium; bar = 500 μm. Arrowheads indicate luminal and glandular epithelium.

Article Snippet: Prevalidated probes and primers for Errfi1 ( Mm00505292_m1 ), Esr1 ( Mm00433149_m1 ), C3 ( Mm01232779_m1 ), Ltf ( Mm00434787_m1 ), Muc1 ( Mm00449604_m1 ), Clca3 ( Mm00489959_m1 ), Pgr ( Mm00435628_m1 ), and 18S rRNA (4319413E) were purchased from Applied Biosystems.

Techniques: Expressing, Quantitative RT-PCR, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction

Journal: Biology of Reproduction

Article Title: ERBB Receptor Feedback Inhibitor 1 Regulation of Estrogen Receptor Activity Is Critical for Uterine Implantation in Mice 1

doi: 10.1095/biolreprod.109.081307

Figure Lengend Snippet: The expression pattern of Errfi1 by E2 and P4 in the uterus. Total RNA used for the RT-PCR assays was prepared from wild-type mice that were treated with P4, E2, E2 plus P4, or vehicle (sesame oil [Veh]) for 4 h, 16 h, and 40 h. The results represent the mean ± SEM of three independent RNA sets. *P < 0.05 and **P < 0.01.

Article Snippet: Prevalidated probes and primers for Errfi1 ( Mm00505292_m1 ), Esr1 ( Mm00433149_m1 ), C3 ( Mm01232779_m1 ), Ltf ( Mm00434787_m1 ), Muc1 ( Mm00449604_m1 ), Clca3 ( Mm00489959_m1 ), Pgr ( Mm00435628_m1 ), and 18S rRNA (4319413E) were purchased from Applied Biosystems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

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