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: Molecular oncology

Article Title: Inhibitor of DNA binding-1 is a key regulator of cancer cell vasculogenic mimicry.

doi: 10.1002/1878-0261.70027

Figure Lengend Snippet: Fig. 2. ID1-targeting siRNA attenuates VM by cancer cells in vitro. (A, E, I) Representative images of ID1 protein expression in MDA-MB- 231-LM2, AsPC-1, and BxPC-3 cancer cells with siRNA for non-silencing (siNS) or targeting ID1 (siID1, constructs A–C) and loading control GAPDH. (B, F, J) Bar graphs of immunoblot densitometry quantified from experimental repeats, siID1 (constructs A–C) normalised to siNS controls. Data are expressed as mean SEM from n = 3 (MDA-MB-231-LM2 and BxPC-3) n = 4 (AsPC-1) experiments. ns, not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001 vs siNS, one-way ANOVA. (C, G, K) Representative images of cancer cells undergoing vasculogenic mimicry (VM) with siNS or siID1 knockdown. For each cell line, the top image depicts VM with low magnification (scale bar = 1 mm) and the bottom image is a higher magnification of the selected region. (D, H, L) Quantitation of VM formation by cancer cells. Data are expressed as mean SEM from n = 3 (MDA-MB-231-LM2, n = 5 (AsPC-1), n = 9 (BxPC-3)) individual experiments. ns, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001 vs siNS, one-way ANOVA.

Article Snippet: Id1 knockdown 4T1.13-LucGFP cells Trilencer-27 siRNA duplexes of three different sequences targeting mouse Id1 (siRNA Oligo Duplex (Locus ID 15901; OriGene)) were reconstituted at 20 lM as per the manufacturer’s instructions.

Techniques: In Vitro, Expressing, Construct, Control, Western Blot, Knockdown, Quantitation Assay

Journal: Molecular oncology

Article Title: Inhibitor of DNA binding-1 is a key regulator of cancer cell vasculogenic mimicry.

doi: 10.1002/1878-0261.70027

Figure Lengend Snippet: Fig. 5. Motif discovery within cis-regulatory elements of ID1 responder genes. (A) Screenshot of the UCSC Genome Browser (v438, hg38) of the selected region (blue highlight) of CST3 based on the histone modifications H3K27Ac (“Layered H3K27Ac”), H3K4Me1 (“Layered H3K4Me1”) and H3K4Me3 (“Layered H3K4Me3”), the ENCODE Candidate Cis-Regulatory Elements (“ENCODE cCREs”), and conservation of 100 vertebrate species (“Cons 100 Verts”). (B) Motif discovery was conducted using the sequences of putative cis-regulatory elements of these 18 downregulated genes. (C) CANNTG was identified as a significantly enriched motif, the motif of basic helix–loop–helix factors (D).

Article Snippet: Id1 knockdown 4T1.13-LucGFP cells Trilencer-27 siRNA duplexes of three different sequences targeting mouse Id1 (siRNA Oligo Duplex (Locus ID 15901; OriGene)) were reconstituted at 20 lM as per the manufacturer’s instructions.

Techniques:

Journal: Molecular oncology

Article Title: Inhibitor of DNA binding-1 is a key regulator of cancer cell vasculogenic mimicry.

doi: 10.1002/1878-0261.70027

Figure Lengend Snippet: Fig. 2. ID1-targeting siRNA attenuates VM by cancer cells in vitro. (A, E, I) Representative images of ID1 protein expression in MDA-MB- 231-LM2, AsPC-1, and BxPC-3 cancer cells with siRNA for non-silencing (siNS) or targeting ID1 (siID1, constructs A–C) and loading control GAPDH. (B, F, J) Bar graphs of immunoblot densitometry quantified from experimental repeats, siID1 (constructs A–C) normalised to siNS controls. Data are expressed as mean SEM from n = 3 (MDA-MB-231-LM2 and BxPC-3) n = 4 (AsPC-1) experiments. ns, not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001 vs siNS, one-way ANOVA. (C, G, K) Representative images of cancer cells undergoing vasculogenic mimicry (VM) with siNS or siID1 knockdown. For each cell line, the top image depicts VM with low magnification (scale bar = 1 mm) and the bottom image is a higher magnification of the selected region. (D, H, L) Quantitation of VM formation by cancer cells. Data are expressed as mean SEM from n = 3 (MDA-MB-231-LM2, n = 5 (AsPC-1), n = 9 (BxPC-3)) individual experiments. ns, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001 vs siNS, one-way ANOVA.

Article Snippet: Small interfering RNA (siRNA) transient knockdown of ID1 Transient silencing of human ID1 expression was achieved by treating cells with trilencer-27 siRNA duplexes of three different sequences targeting human ID1 (siRNA Oligo Duplex (Locus ID 3397; OriGene, Rockville, MD, USA)).

Techniques: In Vitro, Expressing, Construct, Control, Western Blot, Knockdown, Quantitation Assay