Journal: Nature Communications

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy

doi: 10.1038/s41467-018-07608-w

Figure Lengend Snippet: Seesaw effect between CAV1 and HER2 proteins. a Association between CAV1 and HER2 in gastric (NCIN87, KATOIII), bladder (HT1197, UMUC14, UMUC3), breast (MDAMB231, MCF-7, BT474, SKBR3), pancreatic (BxPc3, MiaPaCa, Suit2) and prostate (LnCap, Pc3) cancer cells. The level of CAV1 ( y -axis) and HER2 ( x -axis) are expressed as the ratio of protein/β-actin control, as determined by western blot analysis (upper panel). The non-parametric one-tailed Spearman test was used to determine the correlation coefficients. R HER2/CAV1 , ratio of HER2-to-CAV1 protein levels as determined by western blot analysis from total extracts (lower panel). b Confocal images of immunofluorescence staining of HER2 in s.c. NCIN87 gastric tumors from athymic nude mice, showing that HER2 does not exhibit a predominant membrane staining. Scale bars: 100 μm and 50 μm (inset). c Confocal images of immunofluorescence staining of HER2 and CAV1 in s.c. NCIN87 gastric tumors from athymic nude mice. HER2 presence at the cell membrane in cells where CAV1 is absent (cell clusters 1 and 3) and HER2 almost absent at the cell membrane in cells where CAV1 is present (cell clusters 2 and 4). Scale bars: 50 μm. d Confocal images and quantification (mean ± S.E.M, n = 3) of immunofluorescence staining of HER2 and CAV1 in human HER2-positive gastric tumors containing high (Patient 1) and low (Patient 2) expression of CAV1. HER2 predominant presence at the cell membrane in CAV1-negative tumors (Patient 2). Scale bars: 50 μm. e Western blot analysis of the distribution of HER2 and CAV1 in caveolae fractions isolated from NCIN87 cells. f Western blot of HER2 and CAV1 in the total lysates of NCIN87 cells after blocking protein synthesis with 80 μg mL −1 CHX for 0, 12, 24, 30, 48, 60, 72, and 96 h. CHX cyclohexamide. g HER2 and CAV1 have similar half-lives. Half-lives calculated after western blot analysis of Fig. 1f. Density of western blot bands was quantified by scanning densitometry with ImageJ software. Half-lives were calculated as the time required for protein decrease to 50% of its initial level (mean ± S.E.M, n = 4)

Article Snippet: Gastric human cancer cell lines NCIN87 and KATOIII, breast cancer cell lines MDA-MB-231, SK-BR-3, MCF-7, and BT474 were purchased from American Type Culture Collection (ATCC).

Techniques: Control, Western Blot, One-tailed Test, Immunofluorescence, Staining, Membrane, Expressing, Isolation, Blocking Assay, Software

Journal: Nature Communications

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy

doi: 10.1038/s41467-018-07608-w

Figure Lengend Snippet: Statin treatment increases tumor uptake and efficacy of trastuzumab. a Representative coronal and MIPs PET images and b biodistribution data at 4, 8, 24, and 48 h p.i. of [ 89 Zr]Zr-DFO-trastuzumab in athymic nude mice bearing s.c. gastric tumors. Lovastatin (8.3 mg kg −1 of mice) was orally administrated 12 h prior and at the same time as the tail vein injection of [ 89 Zr]Zr-DFO-trastuzumab (8.14–10.18 Mbq, 80–100 μg protein). Bars, n = 4 mice per group, mean ± S.E.M. MIPs maximum intensity projection. %ID g −1 , percentage of injected dose per gram. Scale bars: 5 mm. c Uptake (upper panel) and autoradiography (lower panel) of [ 89 Zr]Zr-DFO-trastuzumab in organotypic cultures of non-tumor and tumor human bladder tissues. Organotypic cultures were incubated with 25 μM of lovastatin for 4 h prior and at the same time as the addition of [ 89 Zr]Zr-DFO-trastuzumab. Slices were harvested at 3 h after incubation with [ 89 Zr]Zr-DFO-trastuzumab. Bars, n = 3, mean ± S.E.M, * P < 0.05 based on a Student’s t- test. CPM counts per minute. Scale bars: 500 μm. d Superior in vivo therapeutic efficacy of trastuzumab combined with lovastatin when compared with trastuzumab in nu/nu female mice bearing NCIN87 gastric and BT474 breast xenografts, and NSG mice bearing gastric PDXs ( n ≥ 8 mice per group). Intraperitoneal trastuzumab administration 5 mg kg −1 weekly (during 5 weeks) was started at day 0. Lovastatin (4.15 mg kg −1 of mice) was orally administrated 12 h prior and at the same time as the intraperitoneal injection of trastuzumab. e Ability of trastuzumab to mediate in vitro ADCC as determined with peripheral blood mononuclear cells (PBMCs) from healthy donors, in Scr and CAV1-depleted cancer cells (upper panel). Bars, n = 5, mean ± S.E.M, ** P < 0.01 based on a Student’s t- test. Trastuzumab IC 50 response as determined with engineered Jurkat cells stably expressing the FcγRIIIa receptor, in Scr and CAV1-depleted cancer cells (lower panel)

Article Snippet: Gastric human cancer cell lines NCIN87 and KATOIII, breast cancer cell lines MDA-MB-231, SK-BR-3, MCF-7, and BT474 were purchased from American Type Culture Collection (ATCC).

Techniques: Injection, Autoradiography, Incubation, In Vivo, Drug discovery, In Vitro, Stable Transfection, Expressing

Journal: Cancer Drug Resistance

Article Title: Ivosidenib enhances cisplatin sensitivity in ovarian cancer by reducing cancer cell stemness

doi: 10.20517/cdr.2025.51

Figure Lengend Snippet: Screening of a small molecule compound library shows that Ivosidenib enhances the sensitivity of ovarian cancer cells to cisplatin. (A) Breakdown of the compound library based on the molecular pathways associated with the compound targets. (B) Dose-response curves for SK-3rd and SKDDP treated with cisplatin for 72 h. (C) Schematic of the compound screening.(D) Scatter plot showing cell viability in SK-3rd cells treated with compound library alone or the compound library + cisplatin. The red dots represent agents that resulted in over 90% survival of SK-3rd cells alone but less than 60% survival when combined with cisplatin ( n = 4). (E) Corresponding compound names, concentrations, and cell viability data of compounds with > 90% cell viability when used alone but < 60% cell viability combined with 10 µM cisplatin.(F) Scatter plot showing cell viability in SKDDP cells treated with the five selected compounds alone or with the compounds plus cisplatin at a concentration of 10 µM ( n = 4).

Article Snippet: For drug treatment, the working concentrations of these drugs were as follows: 20 μM cisplatin (S1166, Selleck) and 10 μM Ivosidenib (1448347-49-6, MedChemExpress).

Techniques: Drug discovery, Concentration Assay

Journal: Cancer Drug Resistance

Article Title: Ivosidenib enhances cisplatin sensitivity in ovarian cancer by reducing cancer cell stemness

doi: 10.20517/cdr.2025.51

Figure Lengend Snippet: Ivosidenib increases the sensitivity of ovarian cancer cells to cisplatin. (A) Dose-response curves of SKOV3, CAOV3, and OVCAR4 detected by MTT assay treated with cisplatin and 0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM Ivosidenib for 72 h. Data represent mean ± SD of three biologically independent experiments. (B) The lC 50 of SKOV3, CAOV3, and OVCAR4 to cisplatin in (A) were calculated by Graph Pad Prism 9.5.0. Data represent mean ± SD of three biologically independent experiments. ns, not significant, *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (C) Dose-response curves of SKOV3, CAOV3, and OVCAR4 were detected by the MTT assay and treated with Ivosidenib after 72 h. Data represent mean ± SD of three biologically independent experiments. (D) Fa-CI plot of combined treatments of cisplatin (0.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) and Ivosidenib (5 μM, 10 μM) of SKOV3, CAOV3, and OVCAR4 cells. The combination index was calculated using CompuSyn software. CI < 1, =1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. CI, Combination index. Fa, Fraction affected. Each score represents data from three biologically independent experiments. (E) CI scores of SKOV3, CAOV3, and OVCAR4 cells treated with Ivosidenib in combination with cisplatin at the indicated concentrations. Each CI score represents data from three biologically independent experiments.

Article Snippet: For drug treatment, the working concentrations of these drugs were as follows: 20 μM cisplatin (S1166, Selleck) and 10 μM Ivosidenib (1448347-49-6, MedChemExpress).

Techniques: MTT Assay, Software

Journal: Cancer Drug Resistance

Article Title: Ivosidenib enhances cisplatin sensitivity in ovarian cancer by reducing cancer cell stemness

doi: 10.20517/cdr.2025.51

Figure Lengend Snippet: Ivosidenib combined with cisplatin reduces colony formation and enhances apoptosis in ovarian cancer cells. (A) Quantification of cell colony number in SKOV3, CAOV3, and OVCAR4 cells treated with Ivosidenib, cisplatin, or their combination. Data represent mean ± SD of three biologically. ns, not significant, * P < 0.05; *** P < 0.001; **** P < 0.0001 using a two-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (B) Quantification of cell apoptosis analysis in SKOV3, CAOV3, and OVCAR4 cells treated with 10 μM Ivosidenib, 10 μM cisplatin, or their combination for 72 h. Data represent mean ± SD of three biologically. ns, not significant, ** P < 0.01; *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.

Article Snippet: For drug treatment, the working concentrations of these drugs were as follows: 20 μM cisplatin (S1166, Selleck) and 10 μM Ivosidenib (1448347-49-6, MedChemExpress).

Techniques:

Journal: Cancer Drug Resistance

Article Title: Ivosidenib enhances cisplatin sensitivity in ovarian cancer by reducing cancer cell stemness

doi: 10.20517/cdr.2025.51

Figure Lengend Snippet: Ivosidenib increases the sensitivity of SKDDP and SK-3rd to cisplatin. (A) Dose-response curves of SKDDP and SK-3rd detected by MTT assay treated with cisplatin and 0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM Ivosidenib for 72 h. Data represent mean ± SD of three biologically independent experiments. (B) The lC 50 of SKDDP and SK-3rd to cisplatin in (A). Data represent mean ± SD of three biologically independent experiments. ns, not significant, * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (C) Fa-CI plot of combined treatments of cisplatin (0.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) and Ivosidenib (5 μM, 10 μM) of SKDDP, and SK-3rd cells. The combination index was calculated using CompuSyn software. CI < 1, =1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. CI, Combination index. Fa, Fraction affected. Each score represents data from three biologically independent experiments. (D) CI scores of SKDDP and SK-3rd cells treated with Ivosidenib in combination with cisplatin at the indicated concentrations. Each CI score represents data from three biologically independent experiments. (E) Quantification of cell colony number in SKDDP and SK-3rd cells treated with Ivosidenib, cisplatin, or their combination for 72 h. Data represent mean ± SD of three biologically independent experiments. ns, not significant, * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 using a two-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (F) Quantification of cell apoptosis analysis in SKDDP and SK-3rd cells treated with DMSO, 10 μM Ivosidenib, 10 μM cisplatin, or their combination for 72 h. Annexin V-positive cells were analyzed by flow cytometry after treatment. Data represent mean ± SD of three biologically independent experiments. ns, not significant, *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.

Article Snippet: For drug treatment, the working concentrations of these drugs were as follows: 20 μM cisplatin (S1166, Selleck) and 10 μM Ivosidenib (1448347-49-6, MedChemExpress).

Techniques: MTT Assay, Software, Flow Cytometry

Journal: Cancer Drug Resistance

Article Title: Ivosidenib enhances cisplatin sensitivity in ovarian cancer by reducing cancer cell stemness

doi: 10.20517/cdr.2025.51

Figure Lengend Snippet: Ivosidenib reduces the stemness of ovarian cancer cells. (A) Quantitative analysis of the mRNA levels of stemness-related genes, including ALDH1A1 , CD44 , BIM-1 , and KLF-4 in SKOV3, CAOV3, OVCAR4, SKDDP, and SK-3rd treated with DMSO, 10 μM Ivosidenib, 10 μM cisplatin, or their combination for 72 h by qRT-PCR. Data are represented as mean ± SD of three biologically independent experiments. ns, not significant, * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (B) Representative images of ALDH1+ cells were analyzed using flow cytometry in SKOV3 cells treated with DMSO, 10 μM Ivosidenib, 10 μM cisplatin, or their combination for 72 h. DEAB was used as the negative control for ALDH1 activity. (C) The proportion of ALDH1+ cells was analyzed statistically in SKOV3, CAOV3, OVCAR4, SKDDP, and SK-3rd. Data are represented as mean ± SD of three biologically independent experiments. ns, not significant, * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (D) Representative images and statistical histograms of spheres generated by SKOV3, CAOV3, OVCAR4, SKDDP, and SK-3rd cells treated with DMSO or 10 μM Ivosidenib. Scale bar: 50 μm. * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 using an unpaired Student's t -test.

Article Snippet: For drug treatment, the working concentrations of these drugs were as follows: 20 μM cisplatin (S1166, Selleck) and 10 μM Ivosidenib (1448347-49-6, MedChemExpress).

Techniques: Quantitative RT-PCR, Flow Cytometry, Negative Control, Activity Assay, Generated

Journal: Cancer Drug Resistance

Article Title: Ivosidenib enhances cisplatin sensitivity in ovarian cancer by reducing cancer cell stemness

doi: 10.20517/cdr.2025.51

Figure Lengend Snippet: Ivosidenib increases the sensitivity of ovarian cancer organoids to cisplatin. (A) Representative images of PDO1 from ovarian cancer patient treated with DMSO, 10 μM Ivosidenib, 20 μM Cisplatin, or their combination for 72 h. Scale bar: 50 μm. (B-D) The CellTiter-Glo Luminescence Assay of PDO1, PDO2, and PDO3. Data are represented as mean ± SD. ns, not significant, * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 using a one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.

Article Snippet: For drug treatment, the working concentrations of these drugs were as follows: 20 μM cisplatin (S1166, Selleck) and 10 μM Ivosidenib (1448347-49-6, MedChemExpress).

Techniques: Luminescence Assay

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Primer sequences used for molecular cloning

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Primer sequences used for qRT-PCR

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: NAT10 targets LY6E, which mediates ac4C modifications during SINV infection. ( A ) Validation of candidate genes from the RNAseq data using qRT-PCR in NAT10-KD cells infected with SINV. ( B ) Predicted ac4C modification sites for the LY6E pre-mRNA and mature mRNA. ( C ) IP of 293T cells transfected with the NAT10-Myc plasmid and anti-Myc antibody; enriched LY6E mRNA was analyzed using qRT-PCR; the interactions between the NAT10 and LY6E mRNA were also analyzed. ( D ) ( Upper panel ) Immunoblot of the NAT10 immunoprecipitate in panel C. ( Lower panel ) Agarose gel electrophoresis images of the LY6E amplified using qRT-PCR in panel C. ( E ) After incubating with the anti-ac4C antibody and normal rabbit IgG mixed with protein A/G beads at 4°C for 2 h, respectively, incubation was continued with the NAT10-KD Huh7 cell lysate for 2 h. The bound ac4C-modified RNA was eluted and analyzed using qRT-PCR. ( Left panel ) The ac4C-modified RNA was also analyzed using qRT-PCR. ( Right panel ) Agarose gel electrophoresis images of the LY6E amplified using qRT-PCR. Equal amounts of RNA fragments not subjected to immunoprecipitation were used as the input controls. ( F ) ( Upper panel ) Schematic of the 4xS1m aptamer. ( Lower panel ) WT or ac4C site mutated (C–T mut) LY6E mRNA tagged with 4xS1m aptamer was incubated with cell lysates overexpressing NAT10 and separated via streptavidin-conjugated beads. NAT10 in the cell lysate was pulled down, and the LY6E mRNA was detected using an immunoblot. Cells transfected with vectors were used as negative controls. ( G ) ( Upper panel ) Schematic diagram of the dual-luciferase reporter plasmid pmirGLO. ( Lower panel ) Luciferase activity in the NAT10-KD Huh7 ( I ) or A549 (ii) cells transfected with pmirGLO with the WT or ac4C-modifier-site-mutated (C–T mut) the 3′-UTR of the LY6E mRNA. Firefly luciferase activity was normalized to Renilla luciferase activity. ( H, I ) Stability of LY6E mRNA in NAT10-KD Huh7 ( H ) and A549 ( I ) cells after treatment with actinomycin D (5 µg/mL) was analyzed using qRT-PCR at different time points. ( J ) Ly6E mRNA levels were analyzed in Huh7 cells using qRT-PCR at different time points after 24 h of Remodelin treatment with actinomycin D. Blots were quantified with ImageJ software and normalized to control levels. Data are presented as the means ± SEM ( n = 3). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and NS, not significant (A, E, G, H, I, and J, two-way ANOVA with Bonferroni post-test; C, unpaired Student’s t -tests).

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Infection, Biomarker Discovery, Quantitative RT-PCR, Modification, Transfection, Plasmid Preparation, Western Blot, Agarose Gel Electrophoresis, Amplification, Incubation, Immunoprecipitation, Luciferase, Activity Assay, Software, Control

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Predicted ac4C modification sites in the LY6E mRNA determined using PACES ^a

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Modification, Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Mutations in the predicted ac4C modification sites of the LY6E mRNA ^a , ^b

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Modification, Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: SINV is positively affected by NAT10 as it regulates the stability of the LY6E mRNA. ( A ) qRT-PCR analysis of LY6E mRNA expression in LY6E-KD Huh7 cells. ( B ) qRT-PCR analysis of the SINV RNA expression levels in LY6E-KD Huh7 cells at 24 hpi (MOI = 1). ( C ) Immunoblot analysis of the SINV capsid protein expression in LY6E-KD Huh7 cells at 6, 12, and 24 hpi (MOI = 1). ( D ) Plaque formation assay using the SINV infectious virions obtained from the LY6E-KD Huh7 cell culture medium at 24 hpi (MOI = 1). ( E ) qRT-PCR analysis of the SINV RNA expression levels in LY6E-KD Huh7 cells ectopically expressing LY6E and infected with SINV, 24 hpi (MOI = 1). ( F ) Immunoblot analysis of the SINV capsid protein abundance described in panel ( E ). ( G ) Plaque formation assay using the SINV infectious virions obtained from the culture supernatant described in panel ( E ). ( H ) qRT-PCR analysis of the SINV RNA expression levels in NAT10-KD Huh7 cells ectopically expressing LY6E and infected with SINV, 24 hpi (MOI = 1). ( I ) Immunoblot analysis of the SINV capsid protein abundance as described in panel ( H ). ( J ) Plaque formation assay for the SINV infectious virions obtained from the culture supernatant described in panel ( H ). Blots were quantified with ImageJ software and normalized to control levels. Data are presented as the means ± SEM ( n = 3). * P ≤ 0.05 and *** P ≤ 0.001 (A, B, and D, unpaired Student’s t -tests; E, G, H, and J, one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Western Blot, Plaque Formation Assay, Cell Culture, Infection, Quantitative Proteomics, Software, Control

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Working model showing how the loss of NAT10 reduces alphavirus replication. Alphavirus (SINV) infection upregulates NAT10 in host cells and promotes NAT10-mediated ac4C acetylation of LY6E mRNA transcripts, increasing LY6E expression and enhancing alphavirus replication.

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

doi: 10.1101/051391

Figure Lengend Snippet: (A, B) P2Y2 and P2X5 RNA levels are modulated by infection . HFFs were infected with TB40/E-GFP (MOI=3) or mock infected. RNA and protein samples were collected at various times after infection, transcripts were quantified by qRT-PCR and GAPDH was used as an internal control. Results are shown as fold change compared to mock-infected cells. Data were averaged for two biological replicates and error bars represent SEM (C) P2Y2 and P2X5 proteins are modulated by infection . Proteins were subjected to Western blot analyses using antibodies specific for P2Y2 and P2X5. Actin was monitored as a loading control.

Article Snippet: Western blot analyses were performed using primary antibodies recognizing P2Y2 (H-70; Santa Cruz Biotechnology), P2X5 (Sigma-Aldrich), β-actin-HRP (Abcam), or pUL37x1-specific mouse monoclonal antibody 4B6-B [ ].

Techniques: Infection, Quantitative RT-PCR, Control, Western Blot

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

doi: 10.1101/051391

Figure Lengend Snippet: Untreated or UV-irradiated TB40/E virus was applied to HFFs (MOI=3). Samples were collected at 48 hpi (P2Y2) or 24 hpi (P2X5), and transcripts were quantified by qRT-PCR. GAPDH was assayed as an internal control. Results are shown as fold change compared to untreated control virus condition. Data were averaged between two biological replicates and error bars represent SEM.

Article Snippet: Western blot analyses were performed using primary antibodies recognizing P2Y2 (H-70; Santa Cruz Biotechnology), P2X5 (Sigma-Aldrich), β-actin-HRP (Abcam), or pUL37x1-specific mouse monoclonal antibody 4B6-B [ ].

Techniques: Irradiation, Virus, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

doi: 10.1101/051391

Figure Lengend Snippet: HFFs were transfected with siP2Y2, siP2X5, siENPP4, siENTPD2, or scrambled siRNA (siSc) as a control. After 24 h, cells were infected with TB40/E-GFP virus (MOI=3). (A) siRNA validation . Samples were collected at 48 hpi and qRT-PCR was used to measure levels of P2Y2, P2X5, ENPP4 and ENTPD2 transcripts. GAPDH was used as an internal control. The data are represented as fold change compared to siSc control. The mean fold change for three biological replicates are shown and error bars represent SEM. (B) Effect of siRNA-mediated knockdowns on virus yield at 120 hpi . TCID 50 /ml values were determined and fold change was calculated relative to siSc control. Results show the average fold change from two biological replicates and error bars represent SEM. (C) Virus growth kinetics following siRNA-mediated knockdown . Samples were collected after various time intervals. Viral titers were determined by applying the infectious media to a reporter plate of HFFs and immunostaining for IE1 protein 24 h later. The data are presented as percent of IE 1-positive cells averaged from two biological replicates and error bars represent SEM.

Article Snippet: Western blot analyses were performed using primary antibodies recognizing P2Y2 (H-70; Santa Cruz Biotechnology), P2X5 (Sigma-Aldrich), β-actin-HRP (Abcam), or pUL37x1-specific mouse monoclonal antibody 4B6-B [ ].

Techniques: Transfection, Control, Infection, Virus, Biomarker Discovery, Quantitative RT-PCR, Knockdown, Immunostaining

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

doi: 10.1101/051391

Figure Lengend Snippet: HFFs were transfected with siP2Y2 or siSc as a control. After 24 h, cells were infected with TB40/E-GFP virus (MOI=3) or mock infected. (A) P2Y2 KD reduces intracellular vDNA . At 96 hpi, total DNA was isolated from siSc-or siP2Y2-treated, HCMV-infected cells and the level of intracellular viral DNA was measured by qPCR. (B) P2Y2 KD reduces extracellular vDNA . At 96 hpi, total DNA was isolated from media of siSc-or siP2Y2-treated, HCMV-infected cells and the level of extracellular viral DNA was measured by qPCR. (C) P2Y2 KD does not change the particle-to-infectious unit ratio . The infectivity of virus in media collected at 96 hpi was titered and viral DNA was isolated from virions present in the media and quantified by qPCR to calculate a viral DNA copy number-to-infectious unit ratio. The results are depicted as fold change of this ratio. Data were averaged between two biological replicates and error bars represent SEM.

Article Snippet: Western blot analyses were performed using primary antibodies recognizing P2Y2 (H-70; Santa Cruz Biotechnology), P2X5 (Sigma-Aldrich), β-actin-HRP (Abcam), or pUL37x1-specific mouse monoclonal antibody 4B6-B [ ].

Techniques: Transfection, Control, Infection, Virus, Isolation

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

doi: 10.1101/051391

Figure Lengend Snippet: (A) Effects of kaempferol on Ca 2+ levels in AD wt vs. AD sub UL37x1 infected cells . HFFs were infected with either AD wt or AD sub UL37x1 (MOI=l) or mock infected for 2 h and treated with kaempferol (50 μM) or solvent control. Fluo-4 AM dye was applied at 20 hpi. (B) Effects of kaempferol on Ca 2+ levels in wild type vs. UL37xl overexpressing cells . Wild type HFF (WT HFF) cells or HFF cells overexpressing UL37xl [pLVX-UL37x1(1) and pLVX-UL37xl(2)] were treated with kaempferol (50 μM) or solvent control. Fluo-4 AM dye was applied 1 h later. Pictures of cells were taken using the Operetta High-Content Imaging System and the fluorescent signal was measured using ImageJ software based on 10 cells per experimental arm. Results are presented as the intensity of fluorescent signal in arbitrary units [AU]. (C) P2Y2 and UL37xl protein in UL37xl overexpressing cells . Protein samples were collected from two clones of MRC5 fibroblasts overexpressing UL37xl [pLVX-UL37xl (1) and pLVX-UL37xl (2)] and control cells expressing GFP Proteins were separated on SDS-PAGE and Western blot analysis was performed using antibodies recognizing P2Y2, UL37xl and actin as a loading control.

Article Snippet: Western blot analyses were performed using primary antibodies recognizing P2Y2 (H-70; Santa Cruz Biotechnology), P2X5 (Sigma-Aldrich), β-actin-HRP (Abcam), or pUL37x1-specific mouse monoclonal antibody 4B6-B [ ].

Techniques: Infection, Solvent, Control, Imaging, Software, Clone Assay, Expressing, SDS Page, Western Blot

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

doi: 10.1101/051391

Figure Lengend Snippet: sHFFs were infected with either AD wt or AD sub UL37xl virus (MOI=3) or mock-infected for 2 h. Samples were collected at 24 hpi. ( A ) P2Y2, ( B ) UL54, ( C ) UL123 and ( D ) UL37xl transcript levels were determined by qRT-PCR with GAPDH serving as an internal control. Results are shown as fold change compared to mock-infected or AdwMnfected controls. Data are averaged across three biological replicates and error bars represent SEM.

Article Snippet: Western blot analyses were performed using primary antibodies recognizing P2Y2 (H-70; Santa Cruz Biotechnology), P2X5 (Sigma-Aldrich), β-actin-HRP (Abcam), or pUL37x1-specific mouse monoclonal antibody 4B6-B [ ].

Techniques: Infection, Virus, Quantitative RT-PCR, Control

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: EGR3 upregulates ICOSLG through direct promoter binding in an EGR3-overexpressing t(4;11) SEM cell model (A) qRT-PCR proved the transcriptional upregulation of EGR3 (ΔΔC T = 12.03 ± 0.22) and ICOSLG (ΔΔC T = 3.24 ± 0.16) 48h after the transgene induction of the SEM::EGR3 cell model. (B) Western blotting of 20 μg protein lysate confirmed the upregulation of EGR3 (FLAG-tagged) and ICOSLG on the protein level. SEM::mock showed a slight ICOSLG band owing to the basal expression of ICOSLG on SEM cells. (C and D) Chromatin immunoprecipitation followed by qRT-PCR (C) and sequencing (D) displayed the direct binding of EGR3 towards the ICOSLG promoter area. ChIP-qRT-PCR was analyzed through percent input calculation followed by two-tailed unpaired t tests with Welch’s correction to compare the percent input values of SEM:EGR3 α-FLAG with SEM::EGR3 IgG (p = 0.0082), SEM::mock α-FLAG with SEM::mock IgG (p = 0.4452), and SEM::mock α-FLAG with SEM::EGR3 α-FLAG (p = 0.0063). The location of the primer pair used for ChIP-qRT-PCR is displayed as blue lines in the ICOSLG promoter area and indicated below with triangles accordingly.

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, Chromatin Immunoprecipitation, Sequencing, Two Tailed Test

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: Patient characteristics of the dx cohort (n = 50)

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques:

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: IRX1 , EGR3, and ICOSLG expressions correlate strongly in a cohort of 50 infant patients with t(4;11) pro-B ALL (A) Patient age distribution. Patients of all infant age groups are represented, and no age group is overrepresented. (B–F) Pearson correlation matrix visualizes the Pearson r values (B) and corresponding p values (C) for all correlations tested. The strong positive correlations between the expressions of EGR3 -> IRX1 (D), ICOSLG -> EGR3 (E) and ICOSLG -> IRX1 (F) are obvious mapping each patient sample in the correlation plots.

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques:

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: High ICOSLG expression is associated with inferior EFS in a cohort of 43 infant patients with t(4;11) pro-B ALL (A) Patients of the “no event group” (n = 12) and the “event group” (n = 23) were assigned to their respective ICOSLG transcription levels and divided into an ICOSLG lo and an ICOSLG hi subgroup. (B) Kaplan-Meier curves and log rank test of the patients with ICOSLG lo vs ICOSLG hi visualize the clearly inferior EFS of patients with ICOSLG hi (p = 0.0107). (C) Patients separated by age were assigned to the respective ICOSLG transcription level illustrating that the ICOSLG hi group was composed of patients younger and older than 6 months. (D and E) The ICOSLG hi group (n = 8) of all patients (n = 50) displayed significantly elevated IRX1 (p = 0.0006) (D) and EGR3 (p < 0.0001) (E) transcription levels in two-tailed unpaired t tests.

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques: Expressing, Two Tailed Test

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: Patient characteristics of the rel cohort (n = 18)

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques: Biomarker Discovery

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: High EGR3 and ICOSLG expressions are relapse associated (A) HOXA9 , IRX1 , EGR3, and ICOSLG expression levels are displayed in a heatmap for the dx cohort (patients with event; n = 23) and the rel cohort (n = 18). High 2 −ΔCT values indicating high expression are displayed in red. (B–D) Unpaired t tests indicated significantly higher ICOSLG (p < 0.0001) (B) and EGR3 (p < 0.0001) (C) expression levels of the rel cohort compared to the dx cohort, whereas the HOXA9 and IRX1 expression levels did not significantly differ between both cohorts (p HOXA9 = 0.8374; p IRX1 = 0.2737) (E and D). (F) Principal component analysis among IRX1 , EGR3, and ICOSLG expression data with patients assigned to the rel cohort (green), or to the ICOSLG hi (red) or ICOSLG lo (blue) group of the dx cohort. PC1 represents 68%, PC2 22.2% of total variance, respectively. (G) Heatmap of the PCA confirms the clustering of the ICOSLG hi group at diagnosis with the relapse cohort. High 2 −ΔCT values indicating high transcription levels are displayed in red.

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques: Expressing, Biomarker Discovery

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: ICOSLG upregulation in SEM::EGR3 cells leads to the rapid development of T regs upon co-culture with primary T-cells (A) Flow cytometry gating strategy of the co-cultured cells. Cells with low granularity (low SSC) were gated out of all cells. CD3 + cells were selected to subsequently separate CD4 + and CD8 + cells. Viability after co-culture of these populations was assed using Annexin V and 7-AAD staining to ensure that the majority of T-cells were alive throughout co-culture. CD25 + FOXP3 + cells (T regs ) were quantified out of the CD4 + population. (B) Percentages of CD4 + CD25 + FOXP3 + T regs among CD4 + T-cells after co-culturing T-cells from six healthy donors (HD1-HD6) with SEM::mock or SEM::EGR3. SEM::EGR3 co-culture led to 7.96%-23.94% more T regs than SEM::mock co-culture (geometric mean: 1.14 ± 0.009991, p = 0.0023 in a two-tailed ratio paired t test). (C) Replication of the co-culture experiment of HD1-HD3 under the addition of 20 μg/mL mouse IgG1 isotype control or neutralizing monoclonal mouse α-human ICOSLG antibody demonstrated that EGR3-mediated T regs induction could be impaired through ICOSLG antibody blockade. (D and E) ELISAs of the co-culture supernatants depicted that ICOSLG antibody blockade has no effect on the IL-2 level (D) but strongly decreased the IL-10 level (SEM::mock: p = 0.0424; SEM::EGR3: p = 0.0002) (E) of the media. Significance was tested with multiple t tests.

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques: Co-Culture Assay, Flow Cytometry, Cell Culture, Staining, Two Tailed Test, Control

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet: Proposed immune-evasion relapse mechanism in t(4;11) ALL Upper panel: Scheme of ALL chemotherapy. Although complete remission (CR) is frequently achieved after induction therapy, relapse occurs prevalently during consolidation, reinduction, or maintenance phase. Bottom panel: Infant t(4;11) ALL cells characterized by low HOXA transcription overexpress IRX1 , which in turn causes the upregulation of several EGR transcription factors. EGR3 was shown to cause the transcriptional upregulation of ICOSLG. The interaction of ICOSLG on ALL cells with ICOS on T-cells initiates the development of regulatory T-cells (T regs ). The accumulation of T regs in the bone marrow niche could provide ALL cells with an immune privilege allowing them to acquire therapy resistance and to develop minimal residual disease (relapse).

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques:

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet:

Article Snippet: SEM::EGR3 and SEM::mock were induced with 1 μg/mL Doxycycline 24h prior to co-culture and 20 μg/mL neutralizing monoclonal mouse α-human ICOSLG antibody (RnD Systems®) or mouse IgG1 isotype control (InvitrogenTM) were added 2h prior to co-culture.

Techniques: Recombinant, Flow Cytometry, Neutralization, Control, Functional Assay, Virus, Reverse Transcription, Western Blot, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software, Fluorescence, Membrane, Marker

Journal: eLife

Article Title: Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells

doi: 10.7554/eLife.83870

Figure Lengend Snippet:

Article Snippet: The primary antibodies were as follows: anti-MPI (1:5000, GTX103682, GeneTex), anti-β-actin (1:10,000, 010-27841, FUJIFILM Wako), anti-H2AX (1:2000, 938CT5.1.1, Santa Cruz), anti-phospho-H2AX (Ser139) (γH2AX; 1:5000, JBW301, Millipore), anti-RPA2 (1:5000, 9H8, Santa Cruz), anti-Phospho-RPA2 (S33) (1:10,000, A300-246A, Bethyl), anti-MCM2 (1:10,000, D7G11, Cell Signaling Technology), anti-MCM3 (1:10,000, E-8, Santa Cruz), anti-MCM4 (1:5000, GTX109740, GeneTex), anti-MCM5 (1:5000, GTX33310, GeneTex), anti-MCM6 (1:20,000, H-8, Santa Cruz), anti-MCM7 (1:5000, 141.2, Santa Cruz), anti-CDT1 (1:10,000, ab202067, Abcam), anti-CDC6 (1:1000, ab109315, Abcam), anti-ORC2 (1:10,000, 3G6, Santa Cruz), and anti-CDC45 (1:1000, D7G6, Cell Signaling Technology).

Techniques: Retroviral, Recombinant, Drug discovery, Plasmid Preparation, Expressing, Sequencing, CRISPR, Lactate Dehydrogenase Assay, Cloning, Protease Inhibitor, Software

Journal: Journal of Clinical Investigation

Article Title: Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

doi: 10.1172/jci87583

Figure Lengend Snippet: Figure 1. Copy number gains in E2F1 and E2F3 in human primary liver cancer. (A) Box plots illustrating copy number variations in E2F pathway genes in normal liver and HCC samples using values from the TCGA data- base. The center lines in boxes represent the median. The boxes represent the first and third quartiles, and the whiskers represent the highest and lowest values. *P < 0.001, 2-sided Student’s t test vs. control liver. (B) An alternate view of the box plots shown in A. Levels of copy number varia- tions are shown on the y axis and individual patients on the x axis.

Article Snippet: Antibodies used were α-E2F1 (Santa Cruz Biotechnology Inc., clone C-20), α-E2F3 (Santa Cruz Biotechnology Inc., clone C-18), MYC (Abcam, clone 9132), or normal rabbit IgG (Santa Cruz Biotechnology Inc.).

Techniques: Control

Journal: Journal of Clinical Investigation

Article Title: Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

doi: 10.1172/jci87583

Figure Lengend Snippet: Figure 2. Ablation of E2f1 or E2f3b, but not E2f3a, decreases HCC severity in mice. (A) Box plots showing the ratio of liver vs. body weight (liver/body wt.) of 9-month-old WT (E2f+/+) and E2f knockout male mice. Non–DEN-treated cohorts are in white, and DEN-treated are shown in gray. The center lines in boxes represent the median. The box represents the first and third quartiles, and the whiskers represent the high- est and lowest values. Outliers are represented by gray dots. (B) Histopathological classification of mice from A. Carc. MF, carcinoma multifocal; Carc. F, carcinoma focal; Aden, adenoma; FCA, focal cellular atypia; NSL, no significant lesions. Fisher’s exact tests with Bonferroni’s correction for multiple tests. ‡P ≤ 0.001, carcinoma (focal/ multifocal) vs. E2f+/+. (C) Box plots showing liver/body weight of 14-month-old male mice. Non–DEN-treated cohorts are in white, and DEN-treated are shown in gray. *P = 0.040, 1–/– vs. E2f+/+ liver; Wilcoxon method with Bonferroni’s correction. (D) Histopathological classification of mice from C. Fisher’s exact tests with Bonferroni’s correction. ‡P ≤ 0.003, carcinoma vs. E2f+/+.

Article Snippet: Antibodies used were α-E2F1 (Santa Cruz Biotechnology Inc., clone C-20), α-E2F3 (Santa Cruz Biotechnology Inc., clone C-18), MYC (Abcam, clone 9132), or normal rabbit IgG (Santa Cruz Biotechnology Inc.).

Techniques: Knock-Out

Journal: Journal of Clinical Investigation

Article Title: Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

doi: 10.1172/jci87583

Figure Lengend Snippet: Figure 5. Oncogenic functions of E2f1 and E2f3b are cell autonomous. (A) Box plots showing the liver/body weight of 12- to 18-month-old male mice in which the knockin allele or alleles have been deleted in hepatocytes or macrophages using Alb-Cre or Lys-Cre, respectively. Wilcoxon method with Bonferroni’s correction for multiple tests. *P < 0.001 vs. control. (B) Histopathological classification of livers from A. Fisher’s exact tests with Bonferroni’s correction. ‡P = 0.001, carcinoma vs. control. (C) Box plots showing the liver/body weight of 12- to 18-month-old female mice. Wilcoxon method with Bonferroni’s correction. *P < 0.012 vs. control. (D) Histopathological classification of livers from C. Fisher’s exact tests with Bonferroni’s correction. ‡P = 0.001, carcinoma vs. control.

Article Snippet: Antibodies used were α-E2F1 (Santa Cruz Biotechnology Inc., clone C-20), α-E2F3 (Santa Cruz Biotechnology Inc., clone C-18), MYC (Abcam, clone 9132), or normal rabbit IgG (Santa Cruz Biotechnology Inc.).

Techniques: Knock-In, Control

Journal: Journal of Clinical Investigation

Article Title: Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

doi: 10.1172/jci87583

Figure Lengend Snippet: Figure 6. Identification of E2F targets by ChIP sequencing. (A) Immunoblot of protein lysates from MEFs expressing the indicated E2Fs using the pBABE-Hygro (pBH) vector. E2F1 (E2f1 pBH), E2F3A (E2f3a pBH), and E2F3B (E2f3b pBH); 1–/– and 3–/– MEFs are shown as negative controls. GAPDH was used as a loading control. (B) Sequence tag-density heat map showing the distribution of tags for all E2F1, E2F3A, and E2F3B peaks. (C) Percentages of E2F1, E2F3A, and E2F3B peaks in different gene regions. Gene regions were defined by distance from TSS (TSS = 0) as follows: 5′ distal (–50 Gb to –50 kb), 5′ proximal (–50 kb to -2 kb), promoter (–2 kb to +2 kb), gene body (+2kb to end of transcript), 3′ distal (end of transcript to +30 Gb). Number of peaks is indicated above each bar. (D) Graph depicting the number of peaks for E2F1, E2F3A, and E2F3B identified by ChIP-seq and their location relative to the TSS. The promoter region ( ± 2 kb from the TSS) is highlighted in gray, and the top binding motif identified by de novo HOMER within the promoter region is included in color inserts. (E) E2F1, E2F3A, and E2F3B occupancy on selected gene promoters shown at the same scale. (F) Gene ontology using IPA soft- ware depicts the estimated contribution of E2F1, E2F3A, or E2F3B targets to liver hyperplasia and hyperproliferation (left) or HCC-related functions (right). Bars indicate the Benjamini-Hochberg adjusted P value; the threshold of P = 0.05 is shown.

Article Snippet: Antibodies used were α-E2F1 (Santa Cruz Biotechnology Inc., clone C-20), α-E2F3 (Santa Cruz Biotechnology Inc., clone C-18), MYC (Abcam, clone 9132), or normal rabbit IgG (Santa Cruz Biotechnology Inc.).

Techniques: ChIP-sequencing, Western Blot, Expressing, Plasmid Preparation, Control, Sequencing, Binding Assay

Journal: Journal of Clinical Investigation

Article Title: Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

doi: 10.1172/jci87583

Figure Lengend Snippet: Figure 8. Intersection of gene-expression profiling and chromatin binding identifies E2F1 and E2F3B targets. (A) Heat map of Affymetrix microarray data showing differentially expressed genes in 3a1KI/1KI liver tumors when compared with normal liver samples from E2f+/+ and 3a–/– age-matched controls. Differentially expressed genes are defined as having a fold change of 1.5 or more (P ≤ 0.05) relative to 3a–/– samples. (B) Heat map of Affymetrix microarray data showing differentially expressed genes in 3a3bKI/3bKI liver tumors when compared with normal liver samples from E2f+/+ and 3a–/– age-matched controls. Differentially expressed genes are defined as having a fold change of 1.5 or more (P ≤ 0.05) relative to 3a–/– samples. (C) Venn diagram illustrating the overlap of E2F1-specific promoter peaks with upregulated or downregulated genes in 3a1KI/1KI liver tumors identified in A. (D) Venn diagram illustrating the overlap of E2F3B-specific promoter peaks with upregulated or downregulated genes in 3a3bKI/3bKI liver tumors identified in B. (E) Sequence tag-density heat map showing the distribution of E2F1, E2F3A, and E2F3B binding to targets identified in C and D (overlapping groups). (F) ChIP-qPCR validation using E2F1, E2F3, or IgG antibodies in SNU-449 and PLC/PRF5 HCC-derived cells. Occupancy of E2Fs on selected target promoters is shown. A nonpromoter region of TUBA4A (TUBA4A neg) was used as a negative control. Primers were designed to amplify ChIP-seq–identified peak regions.

Article Snippet: Antibodies used were α-E2F1 (Santa Cruz Biotechnology Inc., clone C-20), α-E2F3 (Santa Cruz Biotechnology Inc., clone C-18), MYC (Abcam, clone 9132), or normal rabbit IgG (Santa Cruz Biotechnology Inc.).

Techniques: Gene Expression, Binding Assay, Microarray, Sequencing, ChIP-qPCR, Biomarker Discovery, Derivative Assay, Negative Control, ChIP-sequencing

Journal: Journal of Clinical Investigation

Article Title: Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

doi: 10.1172/jci87583

Figure Lengend Snippet: Figure 9. Association of the expression of E2F1 and E2F3B targets with human HCC. (A) Heat map showing the expression of E2F1 and E2F3B upregulated targets in normal and diseased (cirrhosis, dysplasia, early or advanced HCC) human livers. Genes are grouped based on median expression values per patient group. Genes with the highest median expression in advanced HCC and cirrhosis are denoted on the left. (B) Gene ontology using IPA software showing the esti- mated contribution of E2F1 and E2F3B targets identified in Figure 8 to functions related to cancer, cell cycle, proliferation, fibrosis, and cirrhosis. Bars indicate the Benjamini-Hochberg adjusted P value; the threshold of P = 0.05 is shown. (C) Expression of E2F1 and E2F3B targets in human HCC samples with normal or increased copy numbers of E2F1 and E2F3. E2F1 diploid, 122 samples; gain, 62 samples. E2F3 diploid, 102 samples; gain, 80 samples. Wilcoxon rank sum test Benjamini-Hochberg corrected P values are indicated. (D) Kaplan-Meier plots evaluating the survival time of patients with low to high E2F1/3B target expres- sion. Patients were divided into 3 categories based on target expression. Low expression (0%–5% targets upregulated; black line; n = 136); moderate expression (5%–10% targets upregulated; gray line; n = 113); or high expression (10%–45% targets upregulated; red line; n = 119). log-rank test P values are shown.

Article Snippet: Antibodies used were α-E2F1 (Santa Cruz Biotechnology Inc., clone C-20), α-E2F3 (Santa Cruz Biotechnology Inc., clone C-18), MYC (Abcam, clone 9132), or normal rabbit IgG (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Software

Journal: International Journal of Molecular Sciences

Article Title: Differential Gene Expression in Late-Onset Friedreich Ataxia: A Comparative Transcriptomic Analysis Between Symptomatic and Asymptomatic Sisters

doi: 10.3390/ijms252111615

Figure Lengend Snippet: qRT-PCR analysis for validation of TLR4 , IL20RB , SLITRK5 , TCF21, and GRIN2A mRNA transcripts. Log 2 fold changes determined by RNA-seq and qRT-PCR are presented as mean ± SD. Statistical significance was reached at p < 0.001 for all genes. Three independent fibroblast samples per sister were used ( n = 3).

Article Snippet: According to MIQE guidelines [ ], qRT-PCR was performed using TaqMan Universal Master Mix II (Thermo Fisher Scientific, Waltham, MA, USA) and TaqMan probe assays were performed for TLR4 (assay number Hs00152939_m1, Thermo Fisher Scientific, Waltham, MA, USA), IL20RB (assay number Hs00376373_m1, Thermo Fisher Scientific, Waltham, MA, USA), SLITRK5 (assay number Hs01007362_s1, Thermo Fisher Scientific, Waltham, MA, USA), TCF21 (assay number Hs00162646_m1, Thermo Fisher Scientific, Waltham, MA, USA), and GRIN2A (assay number Hs00168219_m1, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Quantitative RT-PCR, Biomarker Discovery, RNA Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Differential Gene Expression in Late-Onset Friedreich Ataxia: A Comparative Transcriptomic Analysis Between Symptomatic and Asymptomatic Sisters

doi: 10.3390/ijms252111615

Figure Lengend Snippet: Network analysis of the top five genes using Cytoscape v3.10.1 software. The top five genes ( TLR4, SLITRK5, IL20RB , TCF1 , and GRIN2A ) are represented as black circles. All genes involved in the network are indicated as grey circles. Light-brown lines indicate shared protein domains, green lines indicate co-expression, and violet lines indicate genetic interactions.

Article Snippet: According to MIQE guidelines [ ], qRT-PCR was performed using TaqMan Universal Master Mix II (Thermo Fisher Scientific, Waltham, MA, USA) and TaqMan probe assays were performed for TLR4 (assay number Hs00152939_m1, Thermo Fisher Scientific, Waltham, MA, USA), IL20RB (assay number Hs00376373_m1, Thermo Fisher Scientific, Waltham, MA, USA), SLITRK5 (assay number Hs01007362_s1, Thermo Fisher Scientific, Waltham, MA, USA), TCF21 (assay number Hs00162646_m1, Thermo Fisher Scientific, Waltham, MA, USA), and GRIN2A (assay number Hs00168219_m1, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Software, Expressing