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: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitor of Differentiation 1 (ID1) Facilitates the Efficacy of Sorafenib in Non-Small Cell Lung Cancer Cells through Suppressing Epithelial to Mesenchymal Transition

doi: 10.12659/MSM.922148

Figure Lengend Snippet: Sorafenib inhibits ID1 expression. ( A ) Response of cells to sorafenib detected by MTT assay. The average of 3 independent experiments and SD are shown. ( B ) H460 cells were treated with different concentrations of sorafenib. Real-time quantitative PCR was performed to determine the alterations in mRNA expression of ID1, PDGFR, and EGFR. ** P < 0.01; *** P <0.001. ( C ) H460 cells were treated with different concentrations of sorafenib. Western blot experiments were carried out to determine the alteration in ID1 protein expression. ( D ) Immunofluorescence staining for ID1 (red) was conducted in sorafenib-treated H460 cells, and merged images were obtained. DAPI fluorescence is shown in blue. ID1 – inhibitor of differentiation 1; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; SD – standard deviations; PCR – polymerase chain reaction; mRNA – messenger RNA; PDGFR – platelet-derived growth factor receptors; EGFR – epidermal growth factor receptor; DAPI – 4′,6-diamidino-2-phenylindole.

Article Snippet: Chemically synthesized small interfering RNAs (siRNAs) targeting ID1 were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, MTT Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Fluorescence, Polymerase Chain Reaction, Derivative Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitor of Differentiation 1 (ID1) Facilitates the Efficacy of Sorafenib in Non-Small Cell Lung Cancer Cells through Suppressing Epithelial to Mesenchymal Transition

doi: 10.12659/MSM.922148

Figure Lengend Snippet: ID1 downregulation enhances sorafenib efficacy. ( A, B ) H460 cells were transfected with siRNAs targeting ID1. Following treatment with various concentrations of sorafenib for 24 hours ( A ) and 48 hours ( B ), the cell survival rate was determined by MTT assay. ( C, D ) H358 cells were transfected with ID1 overexpression plasmids. Following treatment with various concentrations of sorafenib for 24 hours ( C ) and 48 hours ( D ), the cell survival rate was determined by MTT assay. ID1 – inhibitor of differentiation 1; siRNAs – small interfering RNAs; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Article Snippet: Chemically synthesized small interfering RNAs (siRNAs) targeting ID1 were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, MTT Assay, Over Expression

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitor of Differentiation 1 (ID1) Facilitates the Efficacy of Sorafenib in Non-Small Cell Lung Cancer Cells through Suppressing Epithelial to Mesenchymal Transition

doi: 10.12659/MSM.922148

Figure Lengend Snippet: MG132 inhibits the effect of sorafenib on ID1. ( A ) H460 cells were incubated with various concentrations of sorafenib for 24 hours, with or without pretreatment with MG132. Western blotting was performed to determine the alterations in ID1 expression. ( B, C ) H460 cells transfected with siRNAs targeting ID1 were incubated with various concentrations of sorafenib, with or without pretreatment with MG132. The MTT assay was used to determine the cell survival rate. ID1 – inhibitor of differentiation; siRNAs – small interfering RNAs; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Article Snippet: Chemically synthesized small interfering RNAs (siRNAs) targeting ID1 were purchased from Santa Cruz Biotechnology.

Techniques: Incubation, Western Blot, Expressing, Transfection, MTT Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitor of Differentiation 1 (ID1) Facilitates the Efficacy of Sorafenib in Non-Small Cell Lung Cancer Cells through Suppressing Epithelial to Mesenchymal Transition

doi: 10.12659/MSM.922148

Figure Lengend Snippet: ID1 expression was negatively correlated with EMT biomarkers. ( A ) The pictographs show the morphology of H460, A549, and H358 cells. ( B ) Western blot analysis of EMT markers (E-cadherin and vimentin) and ID1 in H460, A549, and H358 cells. The intensity of the bands was quantified using ImageJ software and normalized to GAPDH ( right panel ). * P <0.05; ** P <0.01; *** P <0.001. ( C ) Immunofluorescence was used to detect the fluorescence intensity of E-cadherin, vimentin and ID1 in H460 and H358 cells. ID1 – inhibitor of differentiation 1; EMT – epithelial to mesenchymal transition.

Article Snippet: Chemically synthesized small interfering RNAs (siRNAs) targeting ID1 were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Software, Immunofluorescence, Fluorescence

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibitor of Differentiation 1 (ID1) Facilitates the Efficacy of Sorafenib in Non-Small Cell Lung Cancer Cells through Suppressing Epithelial to Mesenchymal Transition

doi: 10.12659/MSM.922148

Figure Lengend Snippet: ID1 suppresses EMT in NSCLC. ( A ) Western blot analysis for the expression of EMT-related markers in H460 cells with a negative control and siRNAs targeting ID1 ( left panel ). Western blot analysis for the expression of EMT-related markers in H358 cells with the vector alone as the control and ID1 overexpression plasmids ( right panel ). ( B ) Representative images of wound healing in H460 cells with negative control and siRNAs targeting ID1 are shown in the left panel . Representative images of wound healing in H358 cells with the vector alone as the control and ID1 overexpression plasmids are shown in the right panel . ID1 – inhibitor of differentiation 1; EMT – epithelial to mesenchymal transition; NSCLC – non-small cell lung cancer; siRNAs – small interfering RNAs.

Article Snippet: Chemically synthesized small interfering RNAs (siRNAs) targeting ID1 were purchased from Santa Cruz Biotechnology.

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

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a , b HepG2 cells transfected with siRNA against ID1 (Si-ID1) for 48 h. a RNA was isolated to detect ID1 and p16 to evaluate knockdown efficiency and its impact on p16 mRNA expression. b IL6 concentrations were quantified by ELISA (left). Percentages of SA-β-gal positive cells were calculated (right). c , d Hep3B cells transfected with pcDNA-ID1 for 24 h. c ID1 overexpression efficiency and its impact on p16 mRNA expression were determined by qRT-PCR. d IL6 concentrations were quantified by ELISA (left). Percentages of SA-β-gal positive cells were calculated (right). e A negative correlation between ID1 and p16 mRNA expression was observed in 24 HCC patient samples. f The levels of IL6, which were measured by ELISA, were higher in HCC patients with low concentrations of ID than those with high ones. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with relevant controls (Ctrl) or empty pcDNA

Article Snippet: Chemically synthesized siRNAs targeting ID1 (sc-29356) or p16 (sc-37622) were purchased from Santa Cruz Biotechnology and respectively transfected into HepG2 or Hep3B cells for 48 h using Lipofectamine® 2000 (Invitrogen).

Techniques: Transfection, Isolation, Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a After transfection with si-ID1 for 48 h, MTT assay was employed to detect the efficacy of sorafenib in HepG2 cells with ID1 knockdown. b After transfection with pcDNA-ID1 for 24 h, MTT assay was employed to detect the efficacy of sorafenib in Hep3B cells with ID1overexpression. c After transfection, HepG2 cells were pretreated with neutralizing antibody against IL6 (5 ng/ml) for 2 h prior to co-treatment with sorafenib (5 μM) for 24 h following by MTT assay. d After transfection, Hep3B cells were pretreated with neutralizing antibody against IL6 (5 ng/ml) for 2 h prior to co-treatment with sorafenib (20 μM) for 24 h following by MTT assay. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with relevant control (Ctrl)

Article Snippet: Chemically synthesized siRNAs targeting ID1 (sc-29356) or p16 (sc-37622) were purchased from Santa Cruz Biotechnology and respectively transfected into HepG2 or Hep3B cells for 48 h using Lipofectamine® 2000 (Invitrogen).

Techniques: Transfection, MTT Assay, Knockdown, Control

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: All the recipient mice were divided randomly into two groups ( n = 10 per group). Mice in control group were injected with HepG2-shCtrl cells. Mice in ID1(−) group were injected with HepG2-shID1 cells. Mice were sacrificed after daily administration of sorafenib for 14 days. a Curves of tumor growth in control group (left) and ID1(−) group (right). Green lines indicate the time of the beginning of treatment. b Morphologies of collected tumors in each group (left). Collected tumor volumes were measured by digital caliper and presented as a histogram (right). c Representative mice were monitored by IVIS Imaging System at day 0 and day 14 after sorafenib administration (left). Image intensity was measured and presented as a histogram (right). Tumor tissues were collected for IHC analysis ( d ) and western blot ( e ). The scale bars represent 25 μm. All immunoblots indicate molecular size markers in kDa

Article Snippet: Chemically synthesized siRNAs targeting ID1 (sc-29356) or p16 (sc-37622) were purchased from Santa Cruz Biotechnology and respectively transfected into HepG2 or Hep3B cells for 48 h using Lipofectamine® 2000 (Invitrogen).

Techniques: Control, Injection, Imaging, Western Blot

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a HepG2-SOR1 and HepG2-SOR2 were incubated with sorafenib for 24 h, MTT assay was employed to observe the efficacy. b The expression of ID1, p16, and IL6 mRNA in sorafenib-resistant cell lines was measured by qRT-PCR. c The number of positive SA-β-gal stained cells in sorafenib-resistant cell lines was determined. d Cells were seeded in 6-well plates and cultured for 24 h. Cell supernatant were collected and the secreted IL6 proteins were quantified by ELISA. e Changes of ID1, p16, and p-AKT(473) protein expression in HepG2-SOR1 was detected. f HepG2 cells were incubated with the conditioned medium from HepG2-SOR1 for 24 h. An obvious activation of p-AKT(473) was detected. g HepG2 cells incubated with the supernatant of HepG2 SOR1 were pre-treated with or without IL6 blocking antibody. The difference of sorafenib efficacy in the cells treated with the supernatant alone or the supernatant plus IL6 blocking was confirmed by MTT assay. h 24 h after transfection of pcDNA-ID1 plasmid or empty vector, HepG2 SOR1 cells were pretreated with LY294002 (5 μM) for 1 h prior to co-treatment with sorafenib (20 μM) for 24 h following by MTT assay. Inhibitory rate was analyzed through comparing the average absorbance value in treated cells to control cells. i 24 h after transfection of pcDNA-ID1 plasmid or empty vector, HepG2 SOR1 cells were pretreated with neutralizing antibody against IL6 (5 ng/ml) for 2 h prior to co-treatment with sorafenib (20 μM) for 24 h following by MTT assay. Inhibitory rate was analyzed through comparing the average absorbance value in treated cells to control cells. * p < 0.05; ** p < 0.01; *** p < 0.001. All immunoblots indicate molecular size markers in kDa

Article Snippet: Chemically synthesized siRNAs targeting ID1 (sc-29356) or p16 (sc-37622) were purchased from Santa Cruz Biotechnology and respectively transfected into HepG2 or Hep3B cells for 48 h using Lipofectamine® 2000 (Invitrogen).

Techniques: Incubation, MTT Assay, Expressing, Quantitative RT-PCR, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Blocking Assay, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Stem Cells International

Article Title: Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome

doi: 10.1155/2018/9837035

Figure Lengend Snippet: BMP6 impaired immunomodulatory properties of BMMSCs and downregulated PGE2 via Id1. (a, c, d) Real-time RT-PCR results demonstrated a higher level of Id1 and Id4 mRNA expression in BMMSCs derived from NOD mice compared with BALB/c, while Id2 and Id3 transcripts showed no significant difference between two groups (a) ( ∗∗∗∗ P < 0.0001). (b, e, f) BMP6-treated BALB/c BMMSCs showed an increase in Id1, rather than in Id2, Id3, or Id4 mRNA expression ( ∗∗∗∗ P < 0.0001) Knockdown of Id1 increased BMP6-induced Cox2 (g) and PGE2 (h) downregulation, as evidenced by real-time RT-PCR and ELISA, respectively. ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). (i) CFSE assays indicated that BMP6 inhibited downregulation of T cells proliferation mediated by BMMSCs, while Id1 knockdown attenuated this effect ( ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). N.S.: no significant difference.

Article Snippet: Id1 siRNA (sc-35632, Santa Cruz, USA) and the control scrambled sequence siRNA (sc-36869, Santa Cruz, USA) were transfected using LipofectamineTM 2000 (Invitrogen, USA).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Stem Cells International

Article Title: Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome

doi: 10.1155/2018/9837035

Figure Lengend Snippet: Knockdown of Id1 rescued impaired immunosuppressive capacity of BMMSCs induced by BMP6 in vivo . (a, b) BMMSCs treatment significantly reduced the number of CD4 + T cells, while BMP6-treated BMMSCs showed no significant reduction. When Id1 was blocked by siRNA in BMP6-treated BMMSCs, a greater reduction in CD4 + T cells number was observed ( ∗ P < 0.05, ∗∗∗∗ P < 0.0001). (c, d) A greater ratio of Th1 cells were detected in the presence of BMP6 compared with BMMSCs alone. Knockdown of Id1 significantly decreased the proportion of Th1 cells. ( ∗∗∗∗ P < 0.0001).

Article Snippet: Id1 siRNA (sc-35632, Santa Cruz, USA) and the control scrambled sequence siRNA (sc-36869, Santa Cruz, USA) were transfected using LipofectamineTM 2000 (Invitrogen, USA).

Techniques: Knockdown, In Vivo