guide rna targeting hdac6 exon 5  (Addgene inc)

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 1. HDAC6 knockdown (KD) sensitizes several NSCLC cell lines to ionizing radiation (IR). (A) Smaller fractions of viable cells were found in the A549 HDAC6 KD (HD6 KD) cell line as compared to the A549 control cell line upon IR treatment. Left panel: Western blot confirming HDAC6 knockdown in A549 cells. Right panel: 120 h post-IR, A549 control and HDAC6 stable knockdown cells were suspended in trypan blue. The number of unstained cells (viable), stained cells (non-viable), and total numbers were recorded. Three biological replicates are graphed. Student’s t-tests were performed. * p = 0.0122, ** p = 0.0099, *** p = 0.0021. (B) Smaller fractions of viable cells were found in the H460 HD6 KD cell line as compared to the control cell line upon IR treatment. Left panel: Western blot confirming HDAC6 knockdown in H460 cells. Right panel: H460 stable HDAC6 knockdown cells were either left untreated, or treated with 10 Gy IR. 120 h later, trypan blue staining was conducted as described in (A). Student’s t tests were performed; * p = 0.0154. (C) Smaller fractions of viable cells were

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Knockdown, Control, Western Blot, Staining

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 2. HDAC6 knockdown A549 cells arrest at G2/M phase post-IR. (A) A549 control cells (Ctrl) and A549 HDAC6 stable knockdown cells (HD6 KD) were either left untreated (No Tx, blue histograms) or irradiated with 10 Gy, incubated for 72 h (red histograms), harvested, ethanol fixed, and stained with PI. Cells were then analyzed via flow cytometry. (B) Analysis of the fractions of sub-G1 cells present in the experiments described in (A). Statistical significance was assessed using Student’s t test, with * p < 0.05. (C) Analysis of the cell cycle distribution from the experiments described in (A). (D) A549 control and HDAC6 stable knockdown cells were treated with 10 Gy IR at the indicated time points, and then the cells were stained with immunofluorescence for cyclin A. Results of cyclin A positivity from three biological replicates in these two cell lines were assessed for statistical significance using Student’s t test, with * p < 0.05 and ** p < 0.01. (E) Representative images of the data graphed in (D).

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Knockdown, Control, Irradiation, Incubation, Staining, Cytometry

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 3. Examination of DDR markers in A549 control and A549 HDAC6 knockdown cells post-IR. A549 control and HDAC6 knockdown cells were irradiated with a dose of 10 Gy, harvested at the indicated time points, and the lysates were analyzed via Western blot by a series of antibodies: anti-pATR, anti-pATM, anti-p-p53S15, anti-total p53, and anti-GAPDH in (A) or by anti-pS317Chk1, anti-pS345Chk1, anti-total Chk1, anti-γ-H2AX, anti-acetylated tubulin (ac-tub) and anti-actin in (B). Blots were quantified via ImageJ, and reported quantification was normalized to the signal of the A549 control cells 1 h post-IR. The bar graphs for the expression of indicated DDR proteins are shown in (C–J).

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Control, Knockdown, Irradiation, Western Blot, Expressing

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 4. Examination of DDR markers in A549 control and A549 HDAC6 knockdown cells post-Etoposide treatment. (A) A549 control and HDAC6 knockdown cells were treated with 20 µM Etoposide, harvested at the indicated time points, and the lysates were analyzed via Western blot by a series of antibodies: anti-pS317Chk1, anti-pS345Chk1, anti-total Chk1, anti-γ-H2AX, anti-acetylated tubulin, and anti-GAPDH. Blots were quantified via ImageJ, and reported quantification was normalized to the signal of the A549 control cells 6 h post-Etoposide. The bar graphs for the expression of indicated DDR proteins are shown in (B–E).

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Control, Knockdown, Western Blot, Expressing

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 5. Examination of DDR markers in A549 control and A549 HDAC6 knockdown cells post-Cisplatin treatment. (A) A549 control and HDAC6 knockdown cells were treated with 10 µM Cisplatin, harvested at the indicated time points, and lysates were analyzed via Western blot by a series of antibodies: anti-pS317Chk1, anti-pS345Chk1, anti-total Chk1, anti-γ-H2AX, anti-acetylated tubulin, and anti-GAPDH. Blots were quantified via ImageJ, and reported quantification was normalized to the signal of the A549 control cells 24 h post-Cisplatin treatment, except for the pS317Chk1 bands whose normalization was chosen randomly. The bar graphs for the expression of indicated DDR proteins are shown in (B–E).

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Control, Knockdown, Western Blot, Expressing

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 6. The depletion or inhibition of Chk1 in HDAC6 knockdown A549 cells restores radio-resistance. (A) Establishment of Chk1 knockdown cells in A549 HDAC6 knockdown cells (termed HDAC6KD+Tripz) was described in the Methods. Anti-HDAC6 and anti-Chk1 Western blotting analyses were performed to confirm the efficacy of HDAC6 and Chk1 double knockdown. Anti-pCDC25C, anti-ac-tub, and anti-GAPDH Western blotting analyses were also performed. Representative images of the data graphed Blots were quantified via ImageJ. For HDAC6, the reported quantification was normalized to the signal of the Control group. For the rest of the proteins, the reported quantification was normalized to the signal of the HDAC6 knockdown group. HDAC6KD+Tripz and HDAC6KD cells were plated in triplicate at a concentration of 150 cells/well and treated with the indicated dose of radiation. Cells were incubated for 12 days, fixed with crystal violet. The representative images are shown in (B). The colonies were quantified. Student t test, * p < 0.05, ** p < 0.0008. A bar graph presenting the above colony formation assays is shown in (C). HDAC6KD+Tripz and HDAC6KD cells were treated with 5Gy IR, and the immunofluorescence for Cyclin A was conducted. Representative images are shown in (D). Results of Cyclin A positivity from three experimental replicates, with significance assessed using student’s t test, with * p < 0.01, ** p = 0.0001. A bar graph representing cyclin A positive cells is shown in (E). (F) A549 HDAC6 stable knockdown cells were pre-treated with 0.25 µM of potent Chk1 inhibitor CHIR-124 prior to 10 Gy irradiation. At the indicated time points, cells were harvested and probed for the indicated proteins via Western blot. Blots were quantified via ImageJ, and normalized to the signal of the 0 h timepoint of the HDAC6 knockdowns treated with IR alone.

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Inhibition, Knockdown, Western Blot, Control, Concentration Assay, Incubation, Irradiation

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 7. HDAC6 influences Chk1 protein stability. (A) (From left to right) A549 HDAC6 KO cells generated with the CRISPR-Cas9 system. H157 and H1975 HDAC6 KO cells generated with the CRISPR-Cas9 system. H1299 and A549 inducible HDAC6 knockdown cells (termed H1299i and A549i, respectively) pre-treated with doxycycline for two weeks. Mouse embryonic fibroblasts (MEFs) harvested from age-matched wild-type and HDAC6 KO mice (both from a C57Bl/6 background). Liver, kidney, lung, heart, spleen, and brain tissue harvested from age-matched wild type and transgenic HDAC6 KO mice (both from a C57Bl/6 background). All cell lines and tissues were lysed and analyzed

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Generated, CRISPR, Knockdown, Transgenic Assay

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 8. HDAC6 ubiquitinates Chk1 in vitro and in vivo. (A) HDAC6 ubiquitinates Chk1 in vitro. (A) The in vitro Ub assays were carried out in the presence of E1, E2, Ub, His-Chk1, Flag-HDAC6 in the absence or presence of ATP. The reactions were incubated at 37 ◦C for 2 h, denatured at 95 ◦C for 5 min, then added protein loading buffer. The reactions were loaded into SDS-PAGE followed by Western blotting analysis with the anti-Chk1 antibody. The detailed protocol is described in the Methods and Zhang et al. [22] (B) The Flag-HDAC6 was transfected into 293T cells. The Flag-HDAC6 protein was then isolated anti-Flag M2 agarose followed by Coomassie Blue staining. (C). His-Chk1 was purified from E. coli with Ni-NTA beads followed by Coomassie Blue staining. (D) HDAC6 ubiquitinates Chk1 in vivo. Mammalian expression vectors containing Myc-Chk1, Flag-HDAC6, and His-Ub were transfected into 293T cells. Cells were incubated for 48 h, harvested, and passed through a Ni-NTA column to pull down for His-Ub. Bound proteins were subsequently eluted from the columns, run on an SDS-PAGE gel, and probed for Chk1.

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: In Vitro, In Vivo, Incubation, SDS Page, Western Blot, Transfection, Isolation, Staining, Expressing

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 9. HDAC6 and Chk1 physically interact. (A,B) Mammalian expression vectors containing Flag-Chk1 and HA-HDAC6 were transfected into 293T cells with PEI. 48 h after overexpression, cells were harvested in lysis buffer, incubated with either HA-coated (A) or Flag-coated (B) agarose beads, and the resultant immunoprecipitated protein was run on an SDS-page gel and probed for the reciprocal tag. (C) 293T lysates were probed with anti-Chk1 antibody complexed with protein A/G beads, the beads were washed, and the resulting milieu probed for HDAC6 to detect an endogenous interaction between Chk1 and HDAC6. (D) His-Chk1 was overexpressed in E. coli. His-Chk1 was purified with Ni-NTA agarose beads. Then, GST and GST-HDAC6 were overexpressed in E. coli, and GST-tagged protein was pulled-down and purified by glutathione-agarose. Purified His-Chk1 was incubated with either glutathione agarose-bound GST or GST-HDAC6, and then bound proteins were eluted. The samples were subjected to SDS-PAGE and Western blot analysis.

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Expressing, Transfection, Over Expression, Lysis, Incubation, Immunoprecipitation, SDS Page, Western Blot

Journal: Cells

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

doi: 10.3390/cells9102237

Figure Lengend Snippet: Figure 10. HDAC6 interacts with Chk1 via its DAC1 domain. (A) The indicated Flag-tagged HDAC6 deletion mutant constructs were transfected into 293T cells along with Myc-Chk1. 48 h later, cells were lysed, and lysates were pulled down for Flag. (B) Schematic of the Flag-tagged HDAC6 deletion mutant constructs used for the co-immunoprecipitation in (A). (C) The indicated Myc-tagged Chk1 deletion mutant constructs were transfected into 293T cells along with Flag-HDAC6. 48 h later, cells were lysed, and lysates pulled down for Flag. (D) Schematic of the Myc-tagged Chk1 deletion constructs used for the co-immunoprecipitation in (C).

Article Snippet: Briefly, the guide RNA targeting HDAC6 exon 5 (5′-GAAAGGACACGCAGCGATCT-3′) was selected and constructed into LentiCRISPRv2 vector (Addgene plasmid 52961).

Techniques: Mutagenesis, Construct, Transfection, Immunoprecipitation