Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Effect of cyclin D1 and Id1 on breast cancer cell protein expression and migration . Actively cycling MDA-MB-231 and ZR75-1 cells were monitored 20 h post-transfection with the indicated siRNA (cyclin D1/CDK4/6/Id1) or vector (Id1) for changes in gene or protein expression, and migration. Blots are representative, and plots are mean values from at least three independent experiments. Error bars represent standard deviation. MDA-MB-231 cells: (A) Microarray analysis. Left panel: CCND1 gene expression, right panel: ID1 gene expression (B) Western blot for cyclin d1, Id1 and Actin protein, (C) Cell migration as measured by Boyden chamber assay, dots indicate total number of migrated cells. (D) ChIP assay for Id1 promoter region following cyclin D1 pull down. ZR75-1: (E) Western blot for cyclin d1 and Actin protein (F) Cell migration- Boyden chamber assay. ***P ≥ 0.001, **P ≥ 0.01, *P ≥ 0.05 vs. control, two-tailed student's t-test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Standard Deviation, Microarray, Gene Expression, Western Blot, Boyden Chamber Assay, Control, Two Tailed Test

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Effect of cyclin d1 and Id1 on EMT markers . MDA-MB-231 cells were monitored 20 h post-transfection with the indicated siRNA (cyclin D1/CDK4/6/Id1) or vector (Id1) for changes in EMT-related gene expression by microarray analysis. Additionally, MDA-MB-231 and ZR75-1 gene expression was examined by qPCR assay. Plots are mean values from at least three independent experiments Error bars represent standard deviation. (A) Microarray analysis of SNAI1 , SNAI2 , CDH11 , TWIST1 and VIM gene expression. (B-D) CCND1, ID1 and SNAI2 in MDA-MB-231 cells. (E, F) qPCR analysis of CCND1 and SNAI2 in ZR75-1 cells. ***P ≥ 0.001, **P ≥ 0.01, *P ≥ 0.05 vs. control, two-tailed student's t-test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Transfection, Plasmid Preparation, Gene Expression, Microarray, Standard Deviation, Control, Two Tailed Test

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Distribution of CCND1 and ID1 gene expression according to clinico- pathological parameters in breast cancer patients

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Gene Expression

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Correlation of CCND1 , ID1 , SNAI1 and SNAI2 expression to recurrence free survival . Expression of our genes of interest in relation to recurrence-free survival was examined in a breast cancer database containing 1,107 tumours from Sims et al. (2008). Gene expression intensity was quartiled as 1-low, 2- medium low, 3- medium high and 4- high, and assessed in all patients, ER-positive and ER-negative patients, respectively (A) CCND1 quartiles (B) ID1 quartiles (C) SNAI1 quartiles (D) SNAI2 quartiles. P-value is based on log-rank test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Expressing, Gene Expression

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: EMT-related gene expression intensity and recurrence free survival in CCND1 / ID1 high and low tumours . A breast cancer database was employed to examine (A) Mean-centered average expression of EMT-related genes of interest in CCND1 / ID1 subgroups, and (B, C) Recurrence-free survival of ER-positive and negative patients in CCND1 / ID1 subgroups. P-value is based on log-rank test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Gene Expression, Expressing

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Gene expression of Cyclin d1/Id1 and EMT markers in breast cancer cell lines and claudin-low tumours . Expression of EMT and related genes from the Neve et al cell line (A-C) and Herschkowitz et al. claudin-low (D-F) studies. (A and D) Heatmaps showing relative expression of genes of interest, Red = high, Green = low. (B and E) Scatterplots showing the relationship between breast cancer subgroups and CCND1 / ID1 expression (C and F) Proportion of CCND1- low/ ID1 -high cell lines and tumours in breast cancer subgroups. ***P ≥ 0.001 , Chi 2 test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Gene Expression, Expressing

Journal: Stem Cells International

Article Title: Noggin Combined With Human Dental Pulp Stem Cells to Promote Skeletal Muscle Regeneration

doi: 10.1155/sci/2812390

Figure Lengend Snippet: Noggin antagonize BMP's inhibition of myogenic differentiation of DPSCs. (a) 3D protein structure of Noggin's competitive inhibition of BMP4 binding to BMPRIA. Red indicates Noggin. Gray indicates BMP4. Purple indicates BMPRIA. Cyan indicates a competitive site of Noggin binding to BMPRIA. (b) Protein expression of Pax7, Pax3, Eya2, and Desmin was assessed using a western blot when rescuing with BMP4. (c–e) Protein expression of p-Smad1/5/9 and Smad1 was assessed using western blot by Noggin treatment for different times (3, 6, and 9 h) and subsequently stimulated with BMP4 or control for 1 h (c), and semiquantitative analysis of the expression ratio of (d) p-Smad1/5/9/ β -actin, (e) p-Smad1/5/9/Smad1 were performed ( n = 4). (f, g) Relative mRNAs expression of (f) ID1 and (g) MSX1 were assessed using quantitative PCR ( n = 3). The differences were presented as ⁣ ∗ p < 0.05 and ⁣ ∗∗∗ p < 0.001. 100N = 100 ng/mL Noggin; 200N = 200 ng/mL Noggin. BMP, bone morphogenetic protein; BMPRIA, BMP4 to BMP-receptor I A; DPSCs, dental pulp stem cells; PCR, polymerase chain reaction.

Article Snippet: Antibodies against β -actin (abs830031; Absin Bioscience, Shanghai, China), Pax7 (ab187339), and Myf5 (ab125078) from Abcam; MyHC IIB (BF-F3) and MyHC IIA (SC-71) from Developmental Studies Hybridoma Bank (DSHB, ID, USA); p-Smad 1/5/9 (#13820) and Smad 1 (#6944) from Cell Signaling Technology (CST, MA, USA); and MyoD (#18943), Desmin (#60226), MRF4 (#11754), Pax3 (#21383), Six1 (#10709), Eya2 (#11314), ID1 (#18475), and MSX1 (#17678) from Proteintech (IL, USA) were used.

Techniques: Inhibition, Binding Assay, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: WSS25 Inhibits Growth of Xenografted Hepatocellular Cancer Cells in Nude Mice by Disrupting Angiogenesis via Blocking Bone Morphogenetic Protein (BMP)/Smad/Id1 Signaling

doi: 10.1074/jbc.m110.105544

Figure Lengend Snippet: FIGURE 3. WSS25 potently inhibited the expression of Id1. A, HMEC-1 cells were treated with WSS25 (25 g/ml) for 18 h. RNA was then extracted for the oligo-angiogenesis microarray analysis. Circles indicate Id1. B, WSS25 at 25 g/ml nearly completely inhibited the Id1 mRNA expression in HMEC-1 cells with 18 S rRNA as the internal control. C, WSS25 down-regulated the Id1 protein expression in HMEC-1 cells in a dose-dependent manner. -Actin was used as a control for protein loading. Except for the microarray analysis, all experiments were repeated three times.

Article Snippet: Other antibod- ies used include the Id1 antibody (c20) (Santa Cruz Biotechnology), phosphorylated Smad1/5/8 primary antibody (Cell Signaling Technology), HRP conjugated anti-rabbit and antimouse secondary antibody (Jackson ImmunoResearch Laboratories), anti-CD31 (Boster Biological Technology, Ltd. Wuhan, China), Ki-67 antibody (Abcam, San Francisco, CA), and anti-TUNEL antibody (Trevigen, Inc., Gaithersburg, MD).

Techniques: Expressing, Microarray, Control

Journal: Journal of Biological Chemistry

Article Title: WSS25 Inhibits Growth of Xenografted Hepatocellular Cancer Cells in Nude Mice by Disrupting Angiogenesis via Blocking Bone Morphogenetic Protein (BMP)/Smad/Id1 Signaling

doi: 10.1074/jbc.m110.105544

Figure Lengend Snippet: FIGURE 4. Id1 knockdown in HMEC-1 cells caused disruption of tube for- mationonMatrigel.AandB,HMEC-1cellswereplatedintosix-wellplatesfor 24 h before transfection with Id1 shRNA for 30 h. Both RT-PCR and Western blotting were then used to detect Id1 expression. Id1 shRNA could down- regulate Id1 expression at mRNA level (A) and protein level (B). C, the tube formation of HMEC-1 cells on Matrigel was disrupted (panel c) after Id1 shRNA transfection, compared with the blank (panel a) and sham control (panel b). HMEC-1 cells (2.5 105 cells/well) were seeded into a six-well plate for 24 h before transfection. The shRNA plasmids were transfected into the cells three times 24 h apart using X-fect polymer (Clontech). The cells were used in the tube formation assay as described under “Experimental Procedures” 24 h after the last transfection. Photos were taken after another 12 h of incubation. The results are representative of three experiments.

Article Snippet: Other antibod- ies used include the Id1 antibody (c20) (Santa Cruz Biotechnology), phosphorylated Smad1/5/8 primary antibody (Cell Signaling Technology), HRP conjugated anti-rabbit and antimouse secondary antibody (Jackson ImmunoResearch Laboratories), anti-CD31 (Boster Biological Technology, Ltd. Wuhan, China), Ki-67 antibody (Abcam, San Francisco, CA), and anti-TUNEL antibody (Trevigen, Inc., Gaithersburg, MD).

Techniques: Knockdown, Disruption, Transfection, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Polymer, Tube Formation Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: WSS25 Inhibits Growth of Xenografted Hepatocellular Cancer Cells in Nude Mice by Disrupting Angiogenesis via Blocking Bone Morphogenetic Protein (BMP)/Smad/Id1 Signaling

doi: 10.1074/jbc.m110.105544

Figure Lengend Snippet: FIGURE 5. Effects of WSS25 on Id1, BMP2, and Smad signaling pathway components. A, WSS25 displayed different effects on the expressions of BMP2, BMPRIA, BMPRIB, BMPRII, and Smad4. RT-PCR analysis was performed as described under “Experimental Procedures.” B, WSS25 or Noggin inhibited both Smad1/5/8 phosphorylation and Id1 expression in HMEC-1 cells induced by BMP2. The cells were pretreated with 25 g/ml of WSS25, 5 g/ml noggin or vehicle for 23 h. The cells were then treated with 50 ng/ml of BMP2 or vehicle for another hour. The extracted proteins were analyzed by Western blotting using Id1, Smad1, and pSmad1/5/8 antibodies. -Actin was used as a control for protein loading. The experiments were repeated twice.

Article Snippet: Other antibod- ies used include the Id1 antibody (c20) (Santa Cruz Biotechnology), phosphorylated Smad1/5/8 primary antibody (Cell Signaling Technology), HRP conjugated anti-rabbit and antimouse secondary antibody (Jackson ImmunoResearch Laboratories), anti-CD31 (Boster Biological Technology, Ltd. Wuhan, China), Ki-67 antibody (Abcam, San Francisco, CA), and anti-TUNEL antibody (Trevigen, Inc., Gaithersburg, MD).

Techniques: Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Expressing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: WSS25 Inhibits Growth of Xenografted Hepatocellular Cancer Cells in Nude Mice by Disrupting Angiogenesis via Blocking Bone Morphogenetic Protein (BMP)/Smad/Id1 Signaling

doi: 10.1074/jbc.m110.105544

Figure Lengend Snippet: FIGURE 8. Id1, phosphorylated Smad1/5/8, and CD31 expression levels were lower in WSS25-treated tumortissuethaninthecontrol.Immunohistochemicalanalysiswasperformedontumorsfrommicetreated with 100 mg/kg of WSS25 via tail vein injection or the vehicle as described above. Compared with the control (A), there was also a reduction in the number of blood vessels (shown by reduced expression of the CD31 endothelial marker) in tumors treated with 100 mg/kg WSS25 (B) as indicated by the arrow. Representative images showed that treatment with WSS25 down-regulated Id1 expression in the tumor tissue (C) compared with the control (D) as indicated by the arrow. Phosphorylated Smad1/5/8 expression was also reduced in WSS25-treated tissues (F) compared with the control (E). All images are at 400 magnification.

Article Snippet: Other antibod- ies used include the Id1 antibody (c20) (Santa Cruz Biotechnology), phosphorylated Smad1/5/8 primary antibody (Cell Signaling Technology), HRP conjugated anti-rabbit and antimouse secondary antibody (Jackson ImmunoResearch Laboratories), anti-CD31 (Boster Biological Technology, Ltd. Wuhan, China), Ki-67 antibody (Abcam, San Francisco, CA), and anti-TUNEL antibody (Trevigen, Inc., Gaithersburg, MD).

Techniques: Expressing, Injection, Control, Marker

Journal: International Journal of Medical Sciences

Article Title: Inhibition of USP1 induces apoptosis via ID1/AKT pathway in B-cell acute lymphoblastic leukemia cells

doi: 10.7150/ijms.47597

Figure Lengend Snippet: Effects of SJB3-019A on cell cycle distribution and expression of USP1, ID1 and p-AKT. ( A ) B-ALL cells were incubated with SMI-4a for 24 h, followed by flow cytometry to determine the cell cycle distribution. Data were presented as mean ± SD; *, P < 0.05 versus 0 µM group; and **, P < 0.01 versus 0 µM group. All experiments were performed in triplicate. ( B ) B-ALL cells were treated with SJB3-019A for 24 h, followed by detection of the protein expression of USP1, ID1, AKT and p-AKT using western blot analysis. β-actin was used as a loading control.

Article Snippet: After 12 hours, CCRF-SB and Sup-B15 cells were transfected with synthesized siRNA specifically targeting human USP1 (USP1-siRNA) (TransheepBio, Shanghai, China) or human ID1 (ID1-siRNA) (Santa Cruz) using Lipo6000 Transfection Reagent (Beyotime) according to the manufacturer's procedures.

Techniques: Expressing, Incubation, Flow Cytometry, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Inhibition of USP1 induces apoptosis via ID1/AKT pathway in B-cell acute lymphoblastic leukemia cells

doi: 10.7150/ijms.47597

Figure Lengend Snippet: USP1 regulated the expression of ID1/AKT pathway in B-ALL cells. ( A ) After transfection with USP1-siRNA, the protein expression levels of USP1, ID1, AKT and p-AKT were detected by western blot. Lane 1, Control group; lane 2, NC-siRNA group; lane 3, USP1-siRNA group. ( B ) ID1 expression was analyzed by western blot in B-ALL cells with either NC-siRNA or USP1-siRNA in the presence or absence of MG-132. ( C ) Immunofluorescence staining of ID1 is performed as described in materials and methods. The images shown are under ×1000 magnification. The images are representative of three independent experiments.

Article Snippet: After 12 hours, CCRF-SB and Sup-B15 cells were transfected with synthesized siRNA specifically targeting human USP1 (USP1-siRNA) (TransheepBio, Shanghai, China) or human ID1 (ID1-siRNA) (Santa Cruz) using Lipo6000 Transfection Reagent (Beyotime) according to the manufacturer's procedures.

Techniques: Expressing, Transfection, Western Blot, Control, Immunofluorescence, Staining

Journal: International Journal of Medical Sciences

Article Title: Inhibition of USP1 induces apoptosis via ID1/AKT pathway in B-cell acute lymphoblastic leukemia cells

doi: 10.7150/ijms.47597

Figure Lengend Snippet: SJB3-019A induced B-ALL cell apoptosis partially through ID1-mediated PI3K/AKT pathway. ( A ) real-time PCR analysis of ID1 in B-ALL cells after transfection with either negative control siRNA or siRNA targeting ID1. **, P < 0.01 versus other two group; #, P > 0.05. ( B ) Western blot analysis of the protein expression of ID1, USP1, AKT and p-AKT in B-ALL cells. Lane 1, Control group; lane 2, NC-siRNA group; lane 3, ID1-siRNA group. ( C ) B-ALL cells transfected with either LV-control or LV-ID1 were incubated with SJB3-019A, followed by detection of the intracellular levels of p-AKT using western blot. ( D ) Apoptotic rates of CCRF-SB and Sup-B15 cells after ID1 regulation and SJB3-019A treatment were examined by flow cytometry. Data were presented as mean ± SD; *, P < 0.05, and **, P < 0.01. ( E ) B-ALL cells were infected with LV-control or LV-ID1, and incubated with different concentrations of SJB3-019A, followed by cell viability assessment by CCK-8 assay. *, P < 0.05, **, P < 0.01 versus the control group and LV-control group.

Article Snippet: After 12 hours, CCRF-SB and Sup-B15 cells were transfected with synthesized siRNA specifically targeting human USP1 (USP1-siRNA) (TransheepBio, Shanghai, China) or human ID1 (ID1-siRNA) (Santa Cruz) using Lipo6000 Transfection Reagent (Beyotime) according to the manufacturer's procedures.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot, Expressing, Control, Incubation, Flow Cytometry, Infection, CCK-8 Assay

Journal: Nature communications

Article Title: ID1 expressing macrophages support cancer cell stemness and limit CD8 + T cell infiltration in colorectal cancer.

doi: 10.1038/s41467-023-43548-w

Figure Lengend Snippet: Fig. 3 | ID1-expressing TAMs promote CRC progression partially through inhibiting CD8+ T cell recruitment. a Tumor inhibition rate of MC38 and TAMs mixture s.c. model in the indicated groups, n = 8 mice per group, Welch’s test. b, c Percentage of CD8+ T cells (b), representative mIHC images, and statistical data of CD8+ T cells (c) infiltrated in the tumor tissues as presented in Fig. 2a, n = 4 (b) or 3 (c) mice per group, Student’s t-test, Scale bar, 25 μm. d Representative density dot plots and the statistical data for IFN-γ+ and Granzyme B+ CD8+ T cells in the tumor tissues are presented in Fig. 2a. n = 3 mice per group, Student’s t-test. e Tumor inhibition rate of CT26 spleen-liver metastasis model with i.p. injection of CM from BMDM-derived TAMs in the indicated groups, n = 5 mice per group, Student’s t-test. f Representative mIHC images and statistical data of CD8+ T cells in liver metastatic tumor tissues presented in Fig. 2e, n = 3 mice per group, one-way ANOVA test. Scale bar, 20 μm. g, h Percentage of CD8+ T cells infiltrated in the tumor tissues from

Article Snippet: C57BL/6J mice (male and female, 6-8 weeks old), BALB/c mice (male, 6–8weeks old), BALB/c-Foxn1nu/Nju (named as BALB/c nude hereafter) mice (male, 6 weeks old) were purchased from the BEIJING HFK BIOSCIENCE CO., LTD. OT1 and Lyz2tm1(cre)Ifo/J mice were provided by Cyagen Biosciences Inc. Id1f/f mice were generated by Cyagen Biosciences Inc. Myeloid cell-lineage-specific Id1 deficient mice (Id1Lyz-ko) were generated by crossing Id1f/f mice with Lyz2tm1(cre)Ifo/J mice.

Techniques: Expressing, Inhibition, Injection, Derivative Assay