e2f1 knockeown shrna  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance

doi: 10.1016/j.celrep.2022.111436

Figure Lengend Snippet: The expression of E2F family genes in islets from S961-treated mice versus islets from vehicle-treated mice

Article Snippet: Lentiviral particles for murine E2F1 shRNA , Santa Cruz Biotechnology , Cat#sc-34257-v.

Techniques: Expressing

Journal: Cell reports

Article Title: E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance

doi: 10.1016/j.celrep.2022.111436

Figure Lengend Snippet: (A and C–F) The sera were collected from mice at day 7 of treatment. β cells, mouse islets, or human islets were treated with 20% serum from S-961- or Ns-treated C57BL/6J male mice. (A) MTT assay (absorbance at 570 nm) in control, IRS1KO, IRS2KO, and βIRKO β cells. *p < 0.05, **p < 0.01 (n = 6 biological replicates). (B) Western blot of indicated proteins in scramble- or E2F1-knockdown control β cells. (C) MTT assay in scramble- or E2F1-knockdown control β cells. *p < 0.05, **p < 0.01 (n = 6 biological replicates). (D) Left: representative images of indicated β cells. Nuclei are stained blue, and EdU+ nuclei are stained red. Right: number of ErdU+ β cells. *p < 0.05, **p < 0.01 (n = 6 biological replicates). (E) Mouse islets were incubated with 20% serum from indicated mice in the presence or absence of HLM006474 for 24 h. Left: representative images of islets. Insulin is stained red, nuclei are stained blue, and EdU+ nuclei are stained green. Yellow arrowheads indicate insulin+ and EdU+ cells. Right: number of ErdU+ β cells in the islets. *p < 0.05 (n = 5 mice/group). (F) Human islets from non-diabetes donors were incubated with 20% serum from indicated mice in the presence or absence of HLM006474 for 48 h. Yellow arrowheads indicate insulin+ and EdU+ cells. Left: representative images of islets. Insulin is stained red, nuclei are stained blue, and EdU+ nuclei are stained green. Right: number of ErdU+ β cells in the islets. *p < 0.05 (n = 7 donors). (A and C–F) All data from three or more independent experiments are represented as mean ± SEM. An unpaired two-tailed Student’s t test (A and C), a one-way ANOVA (D and E), and a Mann-Whitney U test (F) were performed. (B) Data are representative of three independent experiments.

Article Snippet: Lentiviral particles for murine E2F1 shRNA , Santa Cruz Biotechnology , Cat#sc-34257-v.

Techniques: MTT Assay, Control, Western Blot, Knockdown, Staining, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance

doi: 10.1016/j.celrep.2022.111436

Figure Lengend Snippet:

Article Snippet: Lentiviral particles for murine E2F1 shRNA , Santa Cruz Biotechnology , Cat#sc-34257-v.

Techniques: Virus, Control, shRNA, Recombinant, Protein Extraction, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Imaging, Microarray, Knock-Out, Software

Journal: Cell reports

Article Title: E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance

doi: 10.1016/j.celrep.2022.111436

Figure Lengend Snippet: The expression of E2F family genes in islets from S961-treated mice versus islets from vehicle-treated mice

Article Snippet: Lentiviral particles for murine E2F1 short hairpin RNA (shRNA) (sc-29297-V) and control scramble shRNA (sc-108080) were purchased from Santa Cruz.

Techniques: Expressing

Journal: Cell reports

Article Title: E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance

doi: 10.1016/j.celrep.2022.111436

Figure Lengend Snippet: (A and C–F) The sera were collected from mice at day 7 of treatment. β cells, mouse islets, or human islets were treated with 20% serum from S-961- or Ns-treated C57BL/6J male mice. (A) MTT assay (absorbance at 570 nm) in control, IRS1KO, IRS2KO, and βIRKO β cells. *p < 0.05, **p < 0.01 (n = 6 biological replicates). (B) Western blot of indicated proteins in scramble- or E2F1-knockdown control β cells. (C) MTT assay in scramble- or E2F1-knockdown control β cells. *p < 0.05, **p < 0.01 (n = 6 biological replicates). (D) Left: representative images of indicated β cells. Nuclei are stained blue, and EdU+ nuclei are stained red. Right: number of ErdU+ β cells. *p < 0.05, **p < 0.01 (n = 6 biological replicates). (E) Mouse islets were incubated with 20% serum from indicated mice in the presence or absence of HLM006474 for 24 h. Left: representative images of islets. Insulin is stained red, nuclei are stained blue, and EdU+ nuclei are stained green. Yellow arrowheads indicate insulin+ and EdU+ cells. Right: number of ErdU+ β cells in the islets. *p < 0.05 (n = 5 mice/group). (F) Human islets from non-diabetes donors were incubated with 20% serum from indicated mice in the presence or absence of HLM006474 for 48 h. Yellow arrowheads indicate insulin+ and EdU+ cells. Left: representative images of islets. Insulin is stained red, nuclei are stained blue, and EdU+ nuclei are stained green. Right: number of ErdU+ β cells in the islets. *p < 0.05 (n = 7 donors). (A and C–F) All data from three or more independent experiments are represented as mean ± SEM. An unpaired two-tailed Student’s t test (A and C), a one-way ANOVA (D and E), and a Mann-Whitney U test (F) were performed. (B) Data are representative of three independent experiments.

Article Snippet: Lentiviral particles for murine E2F1 short hairpin RNA (shRNA) (sc-29297-V) and control scramble shRNA (sc-108080) were purchased from Santa Cruz.

Techniques: MTT Assay, Control, Western Blot, Knockdown, Staining, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance

doi: 10.1016/j.celrep.2022.111436

Figure Lengend Snippet:

Article Snippet: Lentiviral particles for murine E2F1 short hairpin RNA (shRNA) (sc-29297-V) and control scramble shRNA (sc-108080) were purchased from Santa Cruz.

Techniques: Virus, Control, shRNA, Recombinant, Protein Extraction, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Imaging, Microarray, Knock-Out, Software

Journal: Cell death & disease

Article Title: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism.

doi: 10.1038/s41419-022-04798-6

Figure Lengend Snippet: Fig. 1 The NEDD8 pathway is a rational target for HNSCC therapy. A NEDD8 is significantly elevated in clinical cases of HNSCC. Gene expression data from the GDC data portal were analyzed for levels of NEDD8 in 500 HNSCC tumor (T) and 44 adjacent normal (N) tissue samples. Mean ± SD. **Indicates a significant difference at p = 0.0076. B Pevonedistat decreases HNSCC cell viability in a dose-dependent manner. FaDu, A253, Cal27, and Detroit-562 cell lines were treated with varying concentrations of pevonedistat for 72 h. Viability was assessed by MTT assay. Mean ± SD, n = 8. C Pevonedistat stabilizes CRL targets in HNSCC cell lines. FaDu and A253 HNSCC cells were treated with the indicated concentrations of pevonedistat for 24 h. NEDD8, NAE, p21, p27, and γH2AX were measured by immunoblotting. β-Actin was used as a loading control. D Pevonedistat inhibits colony formation in HNSCC cell lines. FaDu and A253 cell lines were plated and treated with various concentrations of pevonedistat for 24 h. The cells were then incubated in fresh media for 10 days and colonies were quantified. Mean ± SD, n = 3.

Article Snippet: ImageJ software was used to calculate the average signal intensity for each condition. shRNA silencing of CUL4A, CUL4B, DDB2, and E2F1 FaDu cells were infected with lentiviral particles containing non-targeted (control) or target-specific short hairpin RNA (shRNA) directed at CUL4A (sc44355-V), CUL4B (sc-37572-V), DDB2 (sc-37799-V), and E2F1 (sc-29297-V) according to the manufacturer’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Gene Expression, MTT Assay, Western Blot, Control, Incubation

Journal: Cell death & disease

Article Title: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism.

doi: 10.1038/s41419-022-04798-6

Figure Lengend Snippet: Fig. 4 Pevonedistat-mediated downregulation of DDB2 drives its synergy with cisplatin. A, B Pevonedistat decreases the expression of DDB2. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat (A) and were treated with 600 nM pevonedistat, 3 μM cisplatin, and the combination (B) for 24 h. DDB2 expression was determined by immunoblotting. C DDB2 was silenced in FaDu cells using lentiviral shRNA. Cells were infected with DDB2 and scramble control shRNA and placed under puromycin selection. DDB2 levels were determined by immunoblotting. D Knockdown of DDB2 increases γH2AX levels following cisplatin treatment. FaDu cells transfected with lentiviral shDDB2 or control particles were treated with 3 μM cisplatin for 24 h. γH2AX signal was observed by fluorescent imaging and quantified using ImageJ software. DAPI was used as a counterstain. Mean signal intensity ±SEM, n = 58 cells per condition. E Knockdown of DDB2 increases cisplatin-induced apoptosis. FaDu cells transfected with lentiviral DDB2 or control shRNA were treated with the indicated concentrations of cisplatin for 48 h. Apoptosis was determined by PI-FACS analysis. Mean ± SD, n = 3. **Indicates a significant difference between indicated groups, p < 0.01.

Article Snippet: ImageJ software was used to calculate the average signal intensity for each condition. shRNA silencing of CUL4A, CUL4B, DDB2, and E2F1 FaDu cells were infected with lentiviral particles containing non-targeted (control) or target-specific short hairpin RNA (shRNA) directed at CUL4A (sc44355-V), CUL4B (sc-37572-V), DDB2 (sc-37799-V), and E2F1 (sc-29297-V) according to the manufacturer’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Western Blot, shRNA, Infection, Control, Selection, Knockdown, Transfection, Imaging, Software

Journal: Cell death & disease

Article Title: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism.

doi: 10.1038/s41419-022-04798-6

Figure Lengend Snippet: Fig. 5 Inhibition of E2F1 underlies pevonedistat-driven suppression of DDB2 levels. A Pevonedistat treatment results in the transcriptional downregulation of DDB2. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat for 24 h. qRT-PCR was used to quantify changes in DDB2 expression. Mean ± SD, n = 3. B Pevonedistat decreases DDB2 promoter activity. FaDu cells were transfected with lentiviral particles containing a DDB2 promoter flanking a luciferase gene. Cells stably expressing this construct were treated with the indicated concentrations of pevonedistat for 24 h and DDB2 promoter activity was quantified. Mean ± SD, n = 3. *Indicates a significant difference from control, p < 0.05. C Pevonedistat increases p21 expression, which is associated with a reduction in Rb phosphorylation. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat for 24 h. Phospho-Rb, total Rb, E2F1, and p21 levels were determined by immunoblotting. D E2F1 knockdown results in decreased DDB2 expression. E2F1 was silenced by lentiviral shRNA. The expression levels of E2F1 and DDB2 were measured by immunoblotting.

Article Snippet: ImageJ software was used to calculate the average signal intensity for each condition. shRNA silencing of CUL4A, CUL4B, DDB2, and E2F1 FaDu cells were infected with lentiviral particles containing non-targeted (control) or target-specific short hairpin RNA (shRNA) directed at CUL4A (sc44355-V), CUL4B (sc-37572-V), DDB2 (sc-37799-V), and E2F1 (sc-29297-V) according to the manufacturer’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Activity Assay, Transfection, Luciferase, Stable Transfection, Construct, Control, Phospho-proteomics, Western Blot, Knockdown, shRNA

Journal: Cell Death & Disease

Article Title: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism

doi: 10.1038/s41419-022-04798-6

Figure Lengend Snippet: A Pevonedistat treatment results in the transcriptional downregulation of DDB2. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat for 24 h. qRT-PCR was used to quantify changes in DDB2 expression. Mean ± SD, n = 3. B Pevonedistat decreases DDB2 promoter activity. FaDu cells were transfected with lentiviral particles containing a DDB2 promoter flanking a luciferase gene. Cells stably expressing this construct were treated with the indicated concentrations of pevonedistat for 24 h and DDB2 promoter activity was quantified. Mean ± SD, n = 3. *Indicates a significant difference from control, p < 0.05. C Pevonedistat increases p21 expression, which is associated with a reduction in Rb phosphorylation. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat for 24 h. Phospho-Rb, total Rb, E2F1, and p21 levels were determined by immunoblotting. D E2F1 knockdown results in decreased DDB2 expression. E2F1 was silenced by lentiviral shRNA. The expression levels of E2F1 and DDB2 were measured by immunoblotting.

Article Snippet: FaDu cells were infected with lentiviral particles containing non-targeted (control) or target-specific short hairpin RNA (shRNA) directed at CUL4A (sc-44355-V), CUL4B (sc-37572-V), DDB2 (sc-37799-V), and E2F1 (sc-29297-V) according to the manufacturer’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Transfection, Luciferase, Stable Transfection, Construct, Control, Phospho-proteomics, Western Blot, Knockdown, shRNA