Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: CHD1L overexpression correlates with worse outcome in cisplatin-treated advanced NSCLC patients and expression of CHD1L in NSCLC cell lines. a Immunohistochemistry showing positive nucleus staining in NSCLC patients for CHD1L, weak staining (left, original magnification, ×200); vs. strong staining (right, original magnification, ×200). The boxed regions are magnified and shown in the panel below (original magnification, ×400); scale bar:10 μm. b High CHD1L expression is correlated with poorer disease-free and overall survival rate in NSCLC patients ( P <0.001). c The levels of CHD1L protein examined by western blotting in six different lung cancer cell lines

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Over Expression, Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: Correlation between the expression of CHD1L and therapy response in NSCLC patients ( N = 30)

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Expressing, Over Expression

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: CHD1L suppresses cisplatin-induced apoptosis in NSCLC cells. a Western blotting reveals that CHD1L was efficiently knocked down by the treatment of CHD1L-shRNA-1 or CHD1L-shRNA-2 in A549 and PC9 cells. b Protein expression of CHD1L in vector, CHD1L-overexpressed (CHD1L) and A549/DDP cells. c Annexin-V-FITC/PI dual staining assay (left, representative plots for flow cytometry; right, bar charts indicating the average percentages of apoptotic cells). d WB assays (c-PARP, cleaved PARP; GAPDH, a loading control). e A549 cells stably transfected with shRNAs specifically targeting CHD1L or control shRNAs were labeled with GFP; meanwhile, the control parental cells were labeled with DsRed. GFP positive and DsRed positive cells were mixed together and subjected to cisplatin treatment (10μM) for 24-72 hours. f Fluorescent images of A549-shCTR-GFP and A549-shCHD1L-GFP cells mixed with A549-DsRed cells before and after cisplatin treatment. g Statistical analysis of flow cytometry data. NC, negative control siRNA; si1, si2, CHD1L siRNAs; Vec, empty vector transfected; CHD1L, CHD1L overexpression; n.s, normal saline. * P <0.05; ** P <0.01; NS, no significance

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Western Blot, shRNA, Expressing, Plasmid Preparation, Staining, Flow Cytometry, Control, Stable Transfection, Transfection, Labeling, Negative Control, Over Expression, Saline

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: Cisplatin resistance is associated with CHD1L activation. a A549 and PC9 cells displayed epithelial morphology, and A549/DDP and PC9/DDP cells exhibited fibroblastic morphology (original magnification, ×200). b Two DDP-resistant cells and their parental cells were treated with indicated concentrations of cisplatin for 48 h and then were subjected to CCK assay (n = 5). The results show that A549/DDP and PC9/DDP cells are more resistant to cisplatin than their parental cells in vitro. c qRT-PCR and Western blotting illustrate increased expression of CHD1L in A549/DDP and PC9/DDP cells. d Schema of step-wise cisplatin treatment on PC9 cells; CHD1L expression during cisplatin treatment was measured using western blotting. Statistics were generated from three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, Student’s t-test; error bar: ±S.D

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Activation Assay, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Generated

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: CHD1L suppresses cisplatin-induced apoptosis in cisplatin-resistance cells. a Western blotting reveals that CHD1L was efficiently knocked down by the treatment of CHD1L-shRNA-1 or CHD1L-shRNA-2 in A549/DDP and PC9/DDP cells. b A549/DDP and PC9/DDP cells were transfected with CHD1L-shRNA-1 or CHD1L-shRNA-2 and seeded in 96-well cell culture plates. The next day, cells were incubated with or without the indicated concentration of cisplatin for 48 h and subsequently subjected to a CCK assay. c WB assays (c-PARP, cleaved PARP) d Annexin-V-FITC/PI dual staining assay (left, representative plots for flow cytometry; right, bar charts indicating the average percentages of apoptotic cells). NC, negative control siRNA; si1, si2, CHD1L siRNAs; n.s, normal saline. * P <0.05; ** P <0.01; NS, no significance

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Western Blot, shRNA, Transfection, Cell Culture, Incubation, Concentration Assay, Staining, Flow Cytometry, Negative Control, Saline

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: Depletion of CHD1L expression enhances the sensitivity of xenograft tumors to cisplatin. a A549/DDP cells transfected with CHD1L-shRNA-1 or CHD1L-shRNA-2 were treated with cisplatin at the indicated concentration for 14 days. Colonies were stained with crystal violet (Left). The number of colonies were taken from three independent experiments (Right). b CHD1L knockdown reverses cisplatin resistance of cisplatin-resistant cells in xenograft tumors implanted onto adult female nude mice. A549/DDP cells transfected with CHD1L-shRNA-1 or CHD1L-shRNA-2 were subcutaneously injected to generate xenograft tumors in nude mice; cisplatin treatment was performed as described in Materials and Methods. Normal saline (n.s.) was used as a treatment control. Images of xenograft tumors harvested at the end of the experiment. c Growth curves of tumor xenografts. d The weights of tumors are presented as a Cleveland dot plot, and the average ±S.D is included (n=6/group; ** P < 0.01; *** P <0.001; NS, no significance)

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Expressing, Transfection, shRNA, Concentration Assay, Staining, Knockdown, Injection, Saline, Control

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: ABCB1 is responsible for CHD1L-induced NSCLC cell cisplatin resistance. a Five genes, ABCB1 , CYP2C19 , SULT1E1 , ERCC3 , and GSTP1 were found to have at least a 2-fold mRNA differential expression in A549-CHD1L cells compared to that in A549-vec using Cancer Drug Resistance RT 2 Profiler™ PCR Array. b Expression of ABCB1, CYP2C19, SULT1E1, ERCC3, and GSTP1 verified in A549-CHD1L and respective control by western blot. c Overexpression of CHD1L and ABCB1 was examined by immunohistochemistry in NSCLC tissues; scale bar, 10 μm, original magnification, ×200. d Silencing of ABCB1 in combination with cisplatin caused a marked inhibition of proliferation in A549-CHD1L cells. e Annexin-V-FITC/PI dual staining assay show that the enhanced cisplatin-resistance ability in A549-CHD1L cells was inhibited by silencing of ABCB1. f Western blot analysis showed that the cisplatin induced γ-H2AX over-expression could be rescued by silencing ABCB1 in CHD1L-overexpressing NSCLC cells. g Images of xenograft tumors harvested at the end of the experiment. h Growth curves of tumor xenografts. i The weights of tumors are presented as a Cleveland dot plot, and the average± S.D. is included (n=6/group; ** P <0.01; *** P <0.001; NS, no significance)

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Quantitative Proteomics, Expressing, Control, Western Blot, Over Expression, Immunohistochemistry, Inhibition, Staining

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: List of genes differentially expressed in A549 cells after CHD1L overexpression using a Cancer Drug Resistance Real-time PCR Array

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Transformation Assay, Activation Assay, Migration, Activity Assay, Functional Assay, Binding Assay

Journal: Cell Death & Disease

Article Title: CHD1L contributes to cisplatin resistance by upregulating the ABCB1–NF-κB axis in human non-small-cell lung cancer

doi: 10.1038/s41419-019-1371-1

Figure Lengend Snippet: ABCB1 upregulation by CHD1L was partly dependent on c-Jun. a Western blot analysis showed that both c-Jun and ABCB1 were increased by enforced CHD1L expression in A549 cells. b Dual luciferase reporter assays show that the increased transcriptional activity and expression levels of ABCB1 by CHD1L were largely blocked after silencing c-Jun in CHD1L-overexpressing NSCLC cells. c The effects on the expression of CHD1L, phosphorylated p65 and phosphorylation of IκBα were analyzed by western blot. Scramble siRNA (siNC) was used as control. d ABCB1 expression and NF-κb activation during cisplatin as depicted in panel. e Schematic diagram depicting a proposed model for a major mechanism of CHD1L and its upregulation in the promotion of NSCLC cell cisplatin-resistance

Article Snippet: The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859).

Techniques: Western Blot, Expressing, Luciferase, Activity Assay, Phospho-proteomics, Control, Activation Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT).

doi: 10.1093/ndt/gfr649

Figure Lengend Snippet: Fig. 1. Identification of CHD1L variants in CAKUT patients. Pedigrees and electropherograms of the three patients with the CHD1L missense variants: CHD1L,c.2098G>A;p.Gly700Arg (exon 18), CHD1L,c.2295A>G;p.Ile765Met (exon 19) and CHD1L,c.2479A>G;p.Ile827Val (exon 21). The affected nucleotide positions in the electropherograms are marked by arrows (A). Genomic location of the three missense variants within the CHD1L gene: CHD1L,c.2098G>A;p.Gly700Arg (exon 18), CHD1L,c.2295A>G;p.Ile765Met (exon 19) and CHD1L,c.2479A>G;p.Ile827Val (exon 21) rela- tive to the functional Snf2 family N-terminal domain (SNF2_N), the helicase superfamily C-terminal domain (Helic_C) and the macro domain (Macro) of the CHD1L protein. Note that all variants are localized close to or within the macro domain (B).

Article Snippet: CHD1L was detected via a primary rabbit anti-CHD1L polyclonal antibody (1:750; Atlas Antibodies) and a secondary goat anti-rabbit Alexa Fluor 568 labelled antibody (1:350; Invitrogen).

Techniques: Functional Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT).

doi: 10.1093/ndt/gfr649

Figure Lengend Snippet: Fig. 2. Quantitative expression of CHD1L mRNA in human fetal and adult tissues. Expression levels were determined by real-time reverse transcription–PCR, normalized to beta-2-microglobulin mRNA expres- sion and displayed relative to the CHD1L mRNA expression in the fetal kidney. CHD1L mRNA expression was the second highest in the fetal kidney compared to the other fetal tissues analysed and was four times as high in fetal compared to adult kidneys. Testis showed the highest CHD1L mRNA expression level of all adult tissues (data not shown).

Article Snippet: CHD1L was detected via a primary rabbit anti-CHD1L polyclonal antibody (1:750; Atlas Antibodies) and a secondary goat anti-rabbit Alexa Fluor 568 labelled antibody (1:350; Invitrogen).

Techniques: Expressing, Reverse Transcription

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT).

doi: 10.1093/ndt/gfr649

Figure Lengend Snippet: Fig. 3. Expression of CHD1L in developing human kidneys. Immunohistochemistry for CHD1L in 8–11 weeks human embryonal kidneys (A–D). All sections were counterstained with haematoxylin; positive immunohistochemical signal is brown; in control samples, 10% FCS was substituted for the primary antibody. Control panel from 8-week-old embryo demonstrating lack of background staining and illustrating early renal development with ureteric bud (u) and stages of nephron formation from comma shaped bodies through developing glomeruli (A). Nearby section demonstrating positive nuclear staining in the nephrogenic zone around the periphery of the kidney, where new nephrons are being formed (B). Control higher power view from 11 weeks showing ureteric bud, ureteric bud tip (t) and developing glomeruli (g), with loose mesenchyme (dotted arrow) and condensed mesenchyme (solid arrow) (C). Nuclear CHD1L was predominantly detected in condensed mesenchyme, although occasional positive cells were detected at the very tips of the ureteric bud (i.e. adjacent to the condensing mesenchyme) and in loose mesenchyme; fully formed glomeruli and deeper, more mature tubule segments were negative (not shown) (D). Immunofluorescence for CHD1L in HEK cells (E–H). CHD1L was stained for using a primary rabbit anti- CHD1L polyclonal antibody and a secondary goat anti-rabbit Alexa Fluor 568 labelled antibody (E), 4’,6-diamidino-2-phenylindole was used as counterstain to delineate the nucleus (F), filamentous actin was visualized in the cytoplasm and at the cell membrane using Alexa488-phalloidin (G). The merged image clearly demonstrates the nuclear localization of CHD1L in embryonal cells (H).

Article Snippet: CHD1L was detected via a primary rabbit anti-CHD1L polyclonal antibody (1:750; Atlas Antibodies) and a secondary goat anti-rabbit Alexa Fluor 568 labelled antibody (1:350; Invitrogen).

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining, Control, Staining, Membrane

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT).

doi: 10.1093/ndt/gfr649

Figure Lengend Snippet: Fig. 4. Detection of megalin, THP and aquaporin-2 in normal human postnatal kidney (left panel) and of CHD1L in healthy and diseased specimens (right panel). Sections were probed with mAbs specific for megalin (A), THP (B), aquaporin-2 (C) and CHD1L (D–F) [normal kidney of 5-year-old (D); hydronephrotic kidney of 16-year-old Patient 2 with hypofunctional CHD1L variant (E); hydroureter from same patient (F)]. Bound antibody was visualized using biotin-labelled secondary antibody in combination with peroxidase-conjugated avidin–biotin complex and 3,3#-diaminobenzidine as substrate. Magnification: 3100 (bar: 200 lm) (A–E), 325 (bar: 1 mm) (F) and 3400 (bar: 50 lm) (inserts in D–F). In normal postnatal sections, CHD1L was expressed in the cytoplasm of tubuloepithelial cells in all segments of the tubule system. CHD1L expression appeared higher in the dysplastic tubules of Patient 2 with a hypofunctional CHD1L variant than in normal kidneys, recapitulating high fetal levels, and was predominantly nuclear in tubular cells and urothelium of hydronephrotic kidney and hydroureter.

Article Snippet: CHD1L was detected via a primary rabbit anti-CHD1L polyclonal antibody (1:750; Atlas Antibodies) and a secondary goat anti-rabbit Alexa Fluor 568 labelled antibody (1:350; Invitrogen).

Techniques: Variant Assay, Avidin-Biotin Assay, Expressing

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT).

doi: 10.1093/ndt/gfr649

Figure Lengend Snippet: Fig. 5. Impairment of mutant CHD1L interaction with PARP1. Untrans- fected, wild-type and mutant CHD1L (Gly700Arg, Ile765Met and Ile827Val) transfected HEK293T cell lysates before (lower two rows) and after IP with c-myc-conjugated agarose beads (upper two rows), probed with anti-c-myc or anti-PARP1 antibodies, respectively (A). Bands detected after IP were quantified using the NIH ImageJ software. The signals measured for the co-precipitated PARP1 were divided by the sig- nals for the immunoprecipitated CHD1L wild-type and mutant proteins. Results are means 6 SEM for three independent experiments. CHD1L and PARP1 interaction is diminished in all three variants (Gly700Arg, Ile765Met and Ile827Val) (B).

Article Snippet: CHD1L was detected via a primary rabbit anti-CHD1L polyclonal antibody (1:750; Atlas Antibodies) and a secondary goat anti-rabbit Alexa Fluor 568 labelled antibody (1:350; Invitrogen).

Techniques: Mutagenesis, Transfection, Software, Immunoprecipitation

Journal: Molecular Cell

Article Title: Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD

doi: 10.1016/j.molcel.2020.12.006

Figure Lengend Snippet:

Article Snippet: ALC1/CHD1L (mouse) , St John’s laboratory , Cat#STJ116477.

Techniques: Binding Assay, Virus, Recombinant, Protease Inhibitor, Purification, Gel Extraction, Fractionation, Western Blot, Mutagenesis, Control, CRISPR, Sequencing, Software

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 1. Isolation of ALC1 from 1q21 amplicon. (A) Amplification of different regions along chromosome 1q in 60 HCC cases was detected by FISH. The number of cases amplified is indicated above each bar. (B) FISH analysis showed that the amplified microdissected DNA probe was specifically hybridized to normal chromosome 1q21 (red signals). This microdissected DNA was used for cDNA selection from an HCC case with 1q21 amplification. (C) ALC1 was mapped to 1q21 by FISH with a BAC clone containing ALC1 (indicated by arrows). (D) A representative exam- ple of ALC1 gene amplification detected in H-4 cells by FISH with the BAC probe (red signals). A BAC probe from 1p32 (green signals) was used as a control. (E) ALC1 was cloned to pEGFP vector. The exogenously expressed ALC1-EGFP fusion protein (green color) was sublocalized in the nucleus. (F) Compared to CHD1, the predicted protein structure of ALC1 also has SNF2_N and HELICc domains.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Isolation, Amplification, Selection, Control, Clone Assay, Plasmid Preparation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 2. Overexpression of ALC1 in primary HCCs and HCC cell lines. (A) A representative example of ALC1 amplification in HCC TMA detected by FISH with the BAC clone contain- ing ALC1. (B) A 98-kD protein was detected by anti-ALC1 antibody. (C) Positive nuclear staining of ALC1 was frequently detected in primary HCC (left) but not in its matched adjacent nontumor tissue specimen (right) by IHC. (D) The IHC result in TMA was verified on a larger tissue section containing HCC tissue (upper part) and surrounding nontumor liver tissue (lower part). RNA expression of ALC1 was tested in (E) primary HCC cases and(F) HCC cell lines by north- ern blot analysis. In primary HCC, ALC1 expression was compared be- tween tumors (T) and their matched nontumor liver tissues (N).

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Over Expression, Staining, RNA Expression, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 3. Oncogenic ability of ALC1. (A) Expression of ALC1 in ALC1- transfected LO2 and QGY-7703 cells detected by northern blot hybrid- ization. Blank vector–transfected cells were used as controls. (B) Rates of colony formation in soft agar detected in ALC1-transfected and blank vector–transfected LO2 and QGY-7703 cells (**P 0.05). (C,D) Rep- resentative examples of tumors formed in nude mice following injection of ALC1-expressing LO2 cells (left) and QGY-7703 cells (right). ALC1- expressing cells and mock cells were injected into the right and left dorsal flanks, respectively. (E) Flow cytometry histogram showing that overex- pression of ALC1 in ALC1-expressing QGY-7703 cells could promote G1/S phase transition compared to vector-transfected QGY-7703 cells.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Expressing, Transfection, Northern Blot, Plasmid Preparation, Injection, Flow Cytometry, Sublimation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 4. Silencing ALC1 expression by siRNA. (A) Two siRNAs (ALC1-si1 and ALC1-si2) could efficiently reduce the expression of ALC1 in H2-M cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. (B) Colony formation ability in soft agar was decreased significantly in siRNA-treated H2-M cells (**P 0.05). (C) Flow cytometry analysis showed that ALC1-si1 could inhibit the cell cycle at the G1/S checkpoint. The percentage of cells in the S phase was decreased from 35% to 23.7%. (D) Western blot analyses indicated that p53 and p21Waf1/Cip1 were down-regulated, whereas cyclin E and Cdk2 were up-regulated in ALC1-transfected QGY-7703 cells in comparison with vector-transfected QGY-7703 cells. -Actin was used as a loading control. (E) Western blot results were quantified by densitometry, and data are presented as mean standard error (n 3). Fold values were first normalized with actin and then compared with vector-transfected QGY-7703 cells.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Western Blot, Transfection, Comparison, Plasmid Preparation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 5. The inhibition role of ALC1 in apoptosis. (A) Representative figures of TUNEL staining images. After cells were treated with STS for 4 hours, more apoptotic cells (bright white) were detected in vector- transfected QGY-7703 cells in comparison with ALC1-transfected 7703 cells. (B) Detection of the apoptotic index between ALC1-transfected and vector-transfected QGY-7703 cells before and after STS treatment (**P 0.05). The data showed that ALC1-transfected QGY-7703 cells could resist STS-induced apoptosis in comparison with QGY-7703 only. (C) Expressions of caspase 3 and Bax were compared between ALC1- transfected and vector-transfected QGY-7703 cells before and after STS treatment by western blot analyses. -Actin was used as a loading control. (D) Protein levels of caspase 3 and Bax were quantified by densitometry, and data are shown as mean standard error (n 3).

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Inhibition, TUNEL Assay, Staining, Plasmid Preparation, Transfection, Comparison, Western Blot, Control