Journal: The Journal of Clinical Investigation

Article Title: Gasdermin C sensitizes tumor cells to PARP inhibitor therapy in cancer models

doi: 10.1172/JCI166841

Figure Lengend Snippet: ( A ) MDA-MB-436 stable cells as indicated were inoculated into the mammary fat pad of nude mice ( n = 10). Mice were administered olaparib. Tumor growth was shown. ( B and C ) 4TO7 cells harboring an empty vector (vector) or expressing WT mouse Gsdmc ( Gsdmc -WT) or the caspase-8 cleavage site D263A mutant ( Gsdmc -mut) were inoculated into the mammary fat pad of nude mice ( B ) or immunocompetent BALB/c mice ( C ) ( n = 10). Mice were administered olaparib. Tumor growth was shown. ( D ) LDH level in tumor slurry of tumors indicated in C was measured. ( E and F ) Same as B and C , except that stable transfectants were established in 4TO7- Brca KO instead of 4TO7 parental cells and injected ( n = 10). ( G ) LDH level in slurry of tumors indicated in F was measured. ( H ) Parental 4TO7 cells mixed with 0%, 15%, or 30% 4TO7- Brca –KO Gsdmc -WT cells were inoculated into BALB/c mice ( n = 10). Mice were administered olaparib. Tumor growth was shown. ( I ) BALB/c mice with 4TO7- Brca –KO Gsdmc -WT or vector tumors were administered olaparib and durable tumor regression was monitored ( n = 10). ( J and K ) 4TO7- Gsdmc -WT ( J ) or 4TO7- Brca –KO Gsdmc -WT ( K ) cells were inoculated into the mammary fat pad of nude mice and immunocompetent BALB/c mice ( n = 10). Mice were administered olaparib. Survival curves were shown. Data represent mean ± SD. 1-way ANOVA was used for A – H . The log-rank test was used for J and K . *** P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting at 1:1,000: α-GSDMC (GTX33979, GeneTex), α-GSDMC (27630-1-AP, Proteintech), α-cleaved caspase-8 (NB100-56116, Novus Biologicals), α-cleaved caspase-6 (9761, Cell Signaling Technology), α-cleaved caspase-3 (NB100-56113, Novus Biologicals), α-cleaved PARP (NB100-56599, Novus Biologicals), α-caspase-8 (4790, Cell Signaling Technology), α-BRCA (NBP1-41185, Novus Biologicals), α-Tubulin (NB100-690, Novus Biologicals), α-Vinculin (4650, Cell Signaling Technology), and α-Flag (F1804, Sigma Aldrich).

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Injection

Journal: The Journal of Clinical Investigation

Article Title: Gasdermin C sensitizes tumor cells to PARP inhibitor therapy in cancer models

doi: 10.1172/JCI166841

Figure Lengend Snippet: ( A ) 4TO7- Brca –KO cells stably expressing an empty vector (vector) or WT mouse Gsdmc ( Gsdmc -WT) or the D263A mutant ( Gsdmc -mut) were inoculated into the mammary fat pad of immunocompetent BALB/c mice ( n = 10). Mice were administered olaparib. Percentage of tumor-infiltrating CD8 + T cells was analyzed. ( B ) Overexpression of eGFP in the stable cells as indicated in A . Then cells and mice were treated same as in A . Mean numbers of eGFP tetramer + (eGFP tet + ) CD8 + T cells per gram of tumor (left). Percentage of IFN-γ + (middle) or TNF-α + (right) CD8 + T cells activated by eGFP peptide. ( C ) Frequency of memory T cell subsets in lymph node (LN), spleen, and tumors of A. Tex, exhausted T cell. ( D ) Initial tumor challenge and mice treatment were same as in A . Tumors were removed on day 18. Then tumor rechallenge of 4TO7 parental cells was performed 60 days after tumor removal. Tumor growth was shown ( n = 10). ( E ) Stable cells as indicated in A were inoculated into the mammary fat pad of immunocompetent BALB/c mice ( n = 10). 4TO7 parental cells were simultaneously injected into contralateral mammary fat pad. Mice were administered olaparib. Tumor growth of 4TO7 parental cells was monitored. ( F and G ) 4TO7- Brca –KO Gsdmc -WT cells were inoculated into BALB/c mice ( n = 10). Mice were administered olaparib. Depletion of CD8 + T cell with anti-CD8. Curves of tumor growth ( F ) and survival ( G ). ( H ) Growth curve of 4TO7- Gsdmc -WT and 4TO7-vector tumors in BALB/c mice ( n = 10) treated with olaparib or PD-1 antibody or the combination. Data represent mean ± SD. 1-way ANOVA was used for A , B , D , E , and H . Unpaired 2-tailed t test was used for F . Log-rank test was used for G . ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting at 1:1,000: α-GSDMC (GTX33979, GeneTex), α-GSDMC (27630-1-AP, Proteintech), α-cleaved caspase-8 (NB100-56116, Novus Biologicals), α-cleaved caspase-6 (9761, Cell Signaling Technology), α-cleaved caspase-3 (NB100-56113, Novus Biologicals), α-cleaved PARP (NB100-56599, Novus Biologicals), α-caspase-8 (4790, Cell Signaling Technology), α-BRCA (NBP1-41185, Novus Biologicals), α-Tubulin (NB100-690, Novus Biologicals), α-Vinculin (4650, Cell Signaling Technology), and α-Flag (F1804, Sigma Aldrich).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Over Expression, Injection

Journal: The Journal of Clinical Investigation

Article Title: Gasdermin C sensitizes tumor cells to PARP inhibitor therapy in cancer models

doi: 10.1172/JCI166841

Figure Lengend Snippet: ( A – F ) MDA-MB-157 and Hs578t cells with deletion of BRCA . Cells were treated with the indicated concentrations of olaparib for 72 hours and subjected to a cell viability assay ( n = 3) ( A and B ). Immunoblotting of GSDMC cleavage in cells treated with the indicated concentrations of olaparib for 72 hours ( C and D ). Cell death measured by LDH release (LDH-released cell death) induced by olaparib at the indicated concentrations ( n = 3) ( E and F ). ( G and H ) 4TO7 parental or Brca -KO cells with ectopic expression of Gsdmc were inoculated into the mammary fat pad of immunocompetent BALB/c mice ( n = 10). Mice were administered olaparib (50 mg/kg) 5 times per week for 18 days. Tumor growth ( G ) and survival ( H ) curves were shown. ( I ) Ectopic expression of Gsdmc in parental or Brca -KO cells of PanO2, MC38, Hepa-1-6, B16. Cells were inoculated into the mammary fat pad of immunocompetent C57BL/6 mice ( n = 10). Mice were treated same as G . Tumor growth curves were shown. Data represent mean ± SD. Unpaired 2-tailed t test was used for A and B . 2-way ANOVA was used for E and F . 1-way ANOVA was used for G and I . Log-rank test was used for H . *** P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting at 1:1,000: α-GSDMC (GTX33979, GeneTex), α-GSDMC (27630-1-AP, Proteintech), α-cleaved caspase-8 (NB100-56116, Novus Biologicals), α-cleaved caspase-6 (9761, Cell Signaling Technology), α-cleaved caspase-3 (NB100-56113, Novus Biologicals), α-cleaved PARP (NB100-56599, Novus Biologicals), α-caspase-8 (4790, Cell Signaling Technology), α-BRCA (NBP1-41185, Novus Biologicals), α-Tubulin (NB100-690, Novus Biologicals), α-Vinculin (4650, Cell Signaling Technology), and α-Flag (F1804, Sigma Aldrich).

Techniques: Viability Assay, Western Blot, Expressing

Journal: International Journal of Nanomedicine

Article Title: EGFR-targeted plasmonic magnetic nanoparticles suppress lung tumor growth by abrogating G2/M cell-cycle arrest and inducing DNA damage

doi: 10.2147/ijn.s65990

Figure Lengend Snippet: Figure 4 Treatment with 225-NP reduced the expression of proteins associated with G2/M checkpoint and DNA repair. Notes: Western blotting showed marked reduction in the expression of BRCA1, Chk1, and phosphorylated Cdc2 and an increase in phosphorylated histone H3 protein at 24 hours in 225-NP-treated cells compared with expression of these proteins in all other treatment groups. β-actin was used as a loading control. Abbreviations: 225-NP, EGFR-targeted hybrid plasmonic magnetic NPs; DNA, deoxyribonucleic acid; EGFR, epidermal growth factor receptor; NP, nanoparticle.

Article Snippet: Primary antibodies against PARP (1:1,000 dilution), phospho-Cdc2 (Tyr15; 1:1,000 dilution), phospho-histone H3 (Ser10; D2C8; 1:1,000 dilution; Cell Signaling Technology, Beverly, MA, USA), anti-phospho/total-EGFR antibody (1:500 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA), phosphohistone H2AX (Ser139) (1:2,500 dilution; EMD Millipore, Billerica, MA, USA), BRCA1 (1:5,000 dilution; Novus, Littleton, CO, USA), Chk1 (G-4), and Cdc2 p34 (1:10,000 dilution; Santa Cruz Biotechnology) were purchased and used.

Techniques: Expressing, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: PALB2 self-interaction controls homologous recombination

doi: 10.1093/nar/gks807

Figure Lengend Snippet: ( A ) Expression of GFP-PALB2-T1 does not affect BRCA1 foci formation. Immunofluorescence staining was performed with anti-BRCA1 (red). The DAPI staining is also shown. ( B ) Purified GST-BRCA1-CC competes with PALB2 self-interaction. Proteins were incubated with PALB2-Flag/His followed by IP with anti-Flag and western blotting with anti-GST and anti-PALB2 antibodies. Left: the order of addition of each protein is shown on the top of the gel. The proteins stained by Coomassie brilliant blue (CBB) are also shown. ( C ) HEK293T cells were transfected with an empty vector or a vector containing Flag-PALB2 ΔT2 , treated with HU (2 mM, 20 h) and Flag-PALB2 ΔT2 was immunoprecipitated with an anti-Flag antibody. Immunoprecipitated proteins were detected by western blotting with the indicated antibodies. Quantification of the increase is estimated by a titration of Flag-PALB2 ΔT2 + HU IP. The band indicated with an asterisk corresponds to the phosphorylated form of PALB2.

Article Snippet: Commercial antibodies used were anti-PALB2 polyclonal antibody (Bethyl), anti-BRCA1 monoclonal antibody (17F8, GeneTex), anti-BRCA1 polyclonal antibody (Millipore), anti-BRCA2 monoclonal antibody (OP95, Calbiochem), anti-RAD51 monoclonal antibody (14B4, Novus), anti-RAD51 polyclonal antibody (Santa-Cruz), anti-Histidine (Clontech), anti-Myc monoclonal antibody (Cell Signaling) and anti-Flag monoclonal antibody (Sigma).

Techniques: Expressing, Immunofluorescence, Staining, Purification, Incubation, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Titration