Journal: Scientific Reports

Article Title: TRIM39 aggravates hepatocellular carcinoma growth through targeting the p62-KEAP1-NRF2 axis

doi: 10.1038/s41598-025-32976-x

Figure Lengend Snippet: p62 is a binding partner of TRIM39. ( A ) Cell lysates from HepG2 cells transfected with empty vector or Flag-TRIM39 were immunoprecipitated with anti-Flag antibody. p62 was identified via mass spectrometry. ( B ) HepG2 cells were co-transfected with Flag-TRIM39 and Myc-p62 for 48 h. Total cell lysates were immunoprecipitated with anti-Flag or anti-Myc antibodies. Myc-p62 and Flag-TRIM39 were detected by western blot. ( C-D ) Immunofluorescence assay of Flag-TRIM39 and Myc-p62 in HepG2 cells. Representative confocal microscopy images were shown. Scale bars, 5 μm. The colocalization of TRIM39 and p62 in HepG2 cells was analyzed. ( E ) The analysis of p62 mRNA levels in TRIM39 stably overexpression or knockdown HCC cells. ( F - G ) The analysis of p62 protein levels in TRIM39 stably overexpression HepG2 and Huh7 cells. *, P < 0.05. ( H - I ) The analysis of p62 protein levels in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ( J ) HepG2 cells transfected with Flag-TRIM39 were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( K ) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( L ) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) and MG132 (10µM) for indicated time and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( M ) TRIM39 stably overexpression and control cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. ( N ) TRIM39 stably knockdown and shNC cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. ( O ) TRIM39 facilitated p62 Ub conjugates by in vitro ubiquitination assay. ( P ) TRIM39 stably overexpression cells were co-transfected with indicated HA-Ub (K-R) mutants for 48 h. Total lysates were immunoprecipitated with anti-p62 antibody. The immunoprecipitation complex was analyzed by western blot with anti-HA and anti-p62 antibodies. ( Q ) TRIM39 stably overexpression cells were transfected with Flag-p62 and its mutants (K420R, K435R). The expression of Flag-p62 was analyzed by western blot.

Article Snippet: Briefly, Purified recombinant His-p62 and GST-TRIM39 proteins were incubated in a mixture of ubiquitin ligation reaction containing 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 4 mM ATP, 1 μg ubiquitin (Yeasen, 20431ES08), 300 ng E1-UBA1 (Targetmol, TMPY-02840), and 500 ng E2-UBE2L3 (Targetmol, TMPY-02842) at 30 °C for 3 h. The reaction was terminated by boiling for 10 min in 2× SDS sample buffer, and the proteins were resolved by SDS-PAGE followed by western blotting with the indicated antibodies.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Western Blot, Immunofluorescence, Confocal, Microscopy, Analysis, Stable Transfection, Over Expression, Knockdown, Control, In Vitro, Ubiquitin Proteomics, Expressing, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Cell-state specific aggresome formation in fibroblasts is driven by stress-activated MAPK signaling

doi: 10.1101/2021.07.23.453501

Figure Lengend Snippet: p38α MAPK and p38β MAPK inhibition with TAK-715 suppresses aggresome formation in fibroblasts. A-B) Average DESeq2 log2(fold change) values for all MAPK cascade or Regulation of stress-activated MAPK cascade genes as proliferating (purple), senescent (blue), quiescent (red) and immortalized (orange) fibroblasts respond to MG132 treatment. C) Proliferating, senescent, quiescent, and immortalized fibroblasts and HEK293T cells were treated with 0.1% DMSO or 10 µM MG132 for 8 hours and then total protein was analyzed by western blot for actin, p62 and P-p62 T269/S272. D-E) Quiescent fibroblasts were treated with 50 µM TAK-715 or 1% DMSO for 24 hours, then additionally treated with 10 µM MG132 for 7 hours still in the presence of TAK-715 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (E). F-G) Immortalized fibroblasts were treated with 10 µM MG132 and either 50 µM TAK-715 or 1% DMSO for 7 hours prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (G). H) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 hours and then total protein was analyzed by western blot for actin, p38 MAPK (p38) and P-p38 MAPK (P-p38; T180/Y182). Aggresomes are denoted by white arrows. Scale bars, 10 µm. **p<0.01, ***p<0.001.

Article Snippet: Western blots probing for p62 (1:1,000; Sigma-Aldrich P0067), P-p62 T269/S272 (1:1,000; PhosphoSolutions p196-269), actin (1:1,000; Bio Rad VMA00048), HSP90β (1:1,000; GeneTex GTX101448), p38 MAPK (1:1,000; Cell Signaling Technology 8690L), P-p38 MAPK (1:1,000; Cell Signaling Technology 4511S) and puromycin (1:1,000; Millipore Sigma MABE343) were performed using the following protocol.

Techniques: Inhibition, Western Blot

Journal: bioRxiv

Article Title: Cell-state specific aggresome formation in fibroblasts is driven by stress-activated MAPK signaling

doi: 10.1101/2021.07.23.453501

Figure Lengend Snippet: TAK1 inhibition supresses aggresome formation in fibroblasts upstream of p38 MAPKs and independent of p62 S351 phosphorylation. A-B) Quiescent fibroblasts were treated with 5 µg/mL 5Z-7-Oxozeaenol or 0.05% DMSO for 24 hours, then additionally treated with 10 µM MG132 for 7 hours still in the presence of TAK-715 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (B). C-D) Immortalized fibroblasts were treated with 10 µM MG132 and either 5 µg/mL 5Z-7-Oxozeaenol or 0.05% DMSO for 7 hours prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (D). E) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 hours and then immunostained for P-p62 S351 (green) and stained for nuclei (Hoechst; blue). F-G) Quiescent fibroblasts were transduced with a vector driving expression of either WT or S351A p62-GFP, treated with 10 µM MG132 for 8 hours and then immunostained for GFP (green) and Hoechst (nuclei; blue). Samples were analyzed for the proportion of cells forming the aggresome (G). H-I) Quiescent fibroblasts were treated with 10 µM MG132 and/or 5 µg/mL 5Z-7-Oxozeaenol for 8 hours as indicated in panel H and then analyzed for expression p38 MAPKs, actin and P-p38 MAPKs (T180/Y182) by western blot. J) A working model for the role of MAPK signaling in aggresome formation in adult mouse dermal fibroblasts. Small red circles depict phosphorylations. Aggresomes are denoted by white arrows. Scale bars, 10 µm. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Western blots probing for p62 (1:1,000; Sigma-Aldrich P0067), P-p62 T269/S272 (1:1,000; PhosphoSolutions p196-269), actin (1:1,000; Bio Rad VMA00048), HSP90β (1:1,000; GeneTex GTX101448), p38 MAPK (1:1,000; Cell Signaling Technology 8690L), P-p38 MAPK (1:1,000; Cell Signaling Technology 4511S) and puromycin (1:1,000; Millipore Sigma MABE343) were performed using the following protocol.

Techniques: Inhibition, Phospho-proteomics, Staining, Transduction, Plasmid Preparation, Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Palmatine treats urticaria by reducing inflammation and increasing autophagy.

doi: 10.3389/fimmu.2023.1268467

Figure Lengend Snippet: FIGURE 6 Immunofluorescence staining (A) and fluorescence intensity (B) of P62. Beclin-1, LC3-I, LC3-II, P62, and GAPDH protein bands in skin samples from the Control, OVA, OVA+LOR and OVA+PAL groups of rats (C). Graphs showing the levels of expression of Beclin-1 (D), P62 (E), LC3-I (F), and LC3-II (G) and LC3-II/LC3-I (H) in the four rat groups. Data are presented as mean ± standard deviation (SD). #P<0.05, ##P<0.01, ###P<0.001 vs. Control group. *P<0.05, ***P<0.001 vs. OVA group. OVA, ovalbumin; LOR, loratadine; PAL, palmatine.

Article Snippet: The membranes were stained with Rexchip Red S dye for 5 min, washed twice with TBST, permeated with TBS, transferred to a sealing solution containing 5% skimmed milk powder in TBST, and sealed by shaking at room temperature for 1 h. The membrane was washed with TBST, sealed, and incubated overnight at 4°C with primary antibody to beclin-1 (1:1000; 66665-1- Ig, Prointech), LC3 A/B (1:1000; 4108, Prointech), P62 (1:5000; 18420- 1-AP, Prointech), LKB1 (1:1000; 3047, Prointech), AMPK (1:1000; 5831, Proteintech), p-AMPK (1:1000), p70S6K1 (1:5000 ratio), pp70S6K1 (1:500; ab32529, Abcam, Cambridge, UK), Atg5-Atg12 complex (1:1000; AAM79 Bio-Rad, CA, USA), or GAPDH (1:1000). frontiersin.org The membranes were washed three times with TBST, followed by incubation at room temperature for 1 h with goat anti-rat IgG H&L (HRP; 1:10,000, ab150157, Abcam) or goat anti-rabbit IgGH&L (HRP; 1:10,000, ab6721, Abcam).

Techniques: Staining, Control, Expressing, Standard Deviation

Journal: Cells

Article Title: Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression

doi: 10.3390/cells13050408

Figure Lengend Snippet: Impact of RUNX2 expression on autophagy markers in melanoma cells. ( a ) Western blots of 1A/1B-light chain 3 (LC3) and p62 in MELHO and in the 1E7, 1B3, and 1F5 RUNX2 KO melanoma cell lines. ( b ) Western blots ( left ) and densitometry analyses ( right ) of LC3 and p62 protein levels in MELHO and in the 1F5 RUNX2 KO cell line mock-treated or treated with bafilomycin A1 or 3-methyladenine. ( c ) Western blots ( left ) and densitometry analyses ( right ) of RUNX2 and LC3 in the 1F5 cell line, in the absence or presence of transfection with the pcDNA3/RUNX2 plasmid at 1.3 µg/mL (RUNX2 ++) or 2.6 µg/mL (RUNX2 +++). ( d ) Western blots ( left ) and densitometry analyses ( right ) of beclin in MELHO and in 1F5 cells without or with transfection with the pcDNA3/RUNX2 plasmid. β-Actin was used as a loading control. Western blot images are representative of three independent experiments. Asterisks indicate statistically significant differences relative to the control (( b , c ), right ) or 1F5 RUNX2 KO melanoma cells ((( d ), right ); *: p < 0.05; **: p < 0.005; ***: p < 0.001). Original blots are presented in .

Article Snippet: The PVDF membranes were probed with specific primary antibodies, to detect RUNX2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 8486), CXCR4 (1:100 dilution; Abcam, Cambridge, UK, reference: ab124824), MMP13 (1:500 dilution; GeneTex, Irvine, CA, USA, reference: GTX 100665), RANKL (1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA, reference: sc377079), LC3B (1:1000 dilution; Invitrogen, Waltham, MA, USA, reference: PA5-22939), p62/SQSTM1 (1:1000 dilution; Rockland, Baltimore, MD, USA, reference: 600-401-HB8), beclin 1 (1:1000 dilution; GeneTex, Irvine, CA, USA, reference: GTX133555), mTOR (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 2983), P-mTOR (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 5536), p70-S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 9202), P-p70-S6 (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 9205), and β-actin (1:10,000 dilution; Thermo Scientific, Waltham, MA, USA, reference: BA3R).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Cell Death & Disease

Article Title: Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer

doi: 10.1038/s41419-023-06344-4

Figure Lengend Snippet: A The label-free quantitative phosphoproteomics was utilized to screen differential proteins. The hierarchical clustering of specific tyrosine sites with high relative phosphorylation of PANC-1 cells after SH2 TrM-(Arg)9 treatment ( n = 3). B Volcano map screening for differential proteins. C Western blot of pY in PDAC cells after SH2 TrM-(Arg)9 incubation. D Western blot of pDDR1(Y792) and DDR1 in PDAC cells upon SH2 TrM-(Arg)9 treatment. E The orthotopic PDAC tumors and the adjacent tissues (AT) from patients with PDAC who underwent surgical resection were subject to IHC analysis with anti-pDDR1(Y792), anti-DDR1 and anti-pY. Scale bar: 200 µm. F Quantification immunostaining of pY, pDDR1(Y792) and DDR1 ( n = 3). ** P < 0.01, *** P < 0.001. G DDR1 kinase (Recombinant human DDR1) activity in the presence and absence of SH2 TrM-(Arg)9 was measured by using an ADP-based phosphatase-coupled kinase assay ( n = 3).*** P < 0.001. H Western blot of pDDR1(Y792), DDR1, pSrc, Src, LC3 and p62 in PDAC cells. I Co-immunoprecipitation between DDR1 and 14-3-3-Beclin1-Akt1 complex with endogenous DDR1 from WCL of PDAC cells treated with SH2 TrM-(Arg)9 or PBS. IgG as control. Cells ( H , I ) under different administrations were pre-treated with collagen (40 µg/mL, 24 h). Representative western blots are shown ( n = 3).

Article Snippet: H , I Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treatment with collagen or PF431396 (T2314, Targetmol, USA) and Selumetinib (T6218, Targetmol, USA).

Techniques: Phospho-proteomics, Western Blot, Incubation, Immunostaining, Recombinant, Activity Assay, Kinase Assay, Immunoprecipitation, Control

Journal: Cell Death & Disease

Article Title: Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer

doi: 10.1038/s41419-023-06344-4

Figure Lengend Snippet: A , B Representative images of autophagosomes or autolysosomes of the PANC-1 cells treated with SH2 TrM-(Arg)9 in nutrient-rich or EBSS starvation conditions. Yellow arrows indicate autophagic structures. C Western blot of LC3, p62, and Beclin1 in PANC-1 cells after SH2 TrM-(Arg)9 incubation in nutrient-rich or EBSS starvation conditions. D , E Representative fluorescence images of GFP-LC3 in PANC-1 cells after SH2 TrM-(Arg)9 or CQ treatment in nutrient-rich or EBSS starvation conditions. F Western blot of LC3, p62, and Beclin1 in PANC-1 cells after SH2 TrM-(Arg)9 or CQ treatment. G , H The immunofluorescence staining of LAMP2 (lysosome-associated membrane protein 2) and LC3. I Co-immunoprecipitation of endogenous Beclin1 with Vps34 and ATG14 from PANC-1 whole-cell lysates (WCL) upon SH2 TrM-(Arg)9 treatment. J Western blot of pmTOR, mTOR, pEIF4EBP1 and EIF4EBP1 in PANC-1 cells after SH2 TrM-(Arg)9 incubation. The data shown are representative of three independent experiments.

Article Snippet: H , I Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treatment with collagen or PF431396 (T2314, Targetmol, USA) and Selumetinib (T6218, Targetmol, USA).

Techniques: Western Blot, Incubation, Fluorescence, Immunofluorescence, Staining, Membrane, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer

doi: 10.1038/s41419-023-06344-4

Figure Lengend Snippet: A Western blot of pDDR1 and DDR1 after siDDR1administration. B Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treated with collagen or siDDR1. C Western blot of pDDR1 and DDR1 after cells were transfected with pcDNA3.1-Flag, pcDNA3.1-Flag-DDR1 (Flag-DDR1), and pcDNA3.1-Flag-DDR1 Y792F (Flag-DDR1 Y792F). D Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treated with collagen or transfected with pcDNA3.1-Flag-DDR1 (Flag-DDR1) and pcDNA3.1-Flag-DDR1 Y792F (Flag-DDR1 Y792F). E Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after being treated with collagen or SH2 TrM-(Arg)9. F , G Representative fluorescence images of GFP-LC3 in PANC-1 cells after SH2 TrM-(Arg)9 or collagen treatment. H , I Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treatment with collagen or PF431396 (T2314, Targetmol, USA) and Selumetinib (T6218, Targetmol, USA). J Co-immunoprecipitation of endogenous STX17 with VAMP8 and SNAP29 from PANC-1 WCL upon SH2 TrM-(Arg)9 treatment. K Co-immunoprecipitation of endogenous STX17 with VAMP8 and SNAP29 from PANC-1 WCL upon siDDR1 treatment. L Co-immunoprecipitation of endogenous STX17 with VAMP8 and SNAP29 from PANC-1 WCL upon pcDNA3.1-Flag-DDR1 Y792F (Flag-DDR1 Y792F) treatment. M Interaction of STX17 and LC3 was affected by SH2 TrM-(Arg)9. IgG as control. Results are representative of three independent experiments with similar results.

Article Snippet: H , I Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treatment with collagen or PF431396 (T2314, Targetmol, USA) and Selumetinib (T6218, Targetmol, USA).

Techniques: Western Blot, Transfection, Fluorescence, Immunoprecipitation, Control

Journal: Cell Death & Disease

Article Title: Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer

doi: 10.1038/s41419-023-06344-4

Figure Lengend Snippet: A , B Changes in cell proliferation between PANC-1 ( A ) and PANC-1/GEM ( B ) (PANC-1-gemcitabine resistant) cells after incubated with gemcitabine (T0251, Targetmol, USA) at different concentrations determined by CCK-8 assay. Cells were treated with SH2 TrM-(Arg)9 (1 μM) plus gemcitabine or gemcitabine-only (GEM) for 4 h. C Changes in cell proliferation between PANC-1 and PANC-1/GEM cells after incubation with GEM for different periods determined by CCK-8 assay. D Effects of SH2 TrM-(Arg)9, GEM, and (GEM + SH2 TrM-(Arg)9) treatments on the proliferation of PANC-1 (left) and PANC-1/GEM (right) cells. Cell viability was measured by CCK-8 assays. E Representative images of colony formation assay showing colonies formed by PANC-1 (up) and PANC-1/GEM (down) cells incubated with SH2 TrM-(Arg)9, GEM, and GEM + SH2 TrM-(Arg)9 treatments. F Bar graph depicting the change in the number of cell colonies. ( n = 3). G Western blot of LC3 and p62 after treated with SH2 TrM-(Arg)9, GEM, and (GEM + SH2 TrM-(Arg)9). H – J Representative fluorescence images of red-only and the yellow puncta in PANC-1 and PANC-1/GEM cells after SH2 TrM-(Arg)9, GEM, and (GEM + SH2 TrM-(Arg)9) treatment. K Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, and ERK1/2 after treated with SH2 TrM-(Arg)9, GEM and (GEM + SH2 TrM-(Arg)9). L Co-immunoprecipitation of STX17 with VAMP8 and SNAP29 from PANC-1/GEM WCL upon SH2 TrM-(Arg)9 treatment. IgG as control. The gemcitabine usage condition of Fig. 5D-K were 4 μM, 24 h. The SH2 TrM-(Arg)9 condition of Fig. 5D-L were 1 μM, 2 h. Results are representative of three independent experiments with similar results. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: H , I Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treatment with collagen or PF431396 (T2314, Targetmol, USA) and Selumetinib (T6218, Targetmol, USA).

Techniques: Incubation, CCK-8 Assay, Colony Assay, Western Blot, Fluorescence, Immunoprecipitation, Control

Journal: Cell Death & Disease

Article Title: Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer

doi: 10.1038/s41419-023-06344-4

Figure Lengend Snippet: A Excised tumors of PANC-1 and PANC-1/GEM xenografts mouse models in different groups are shown. Mice were injected with different treatments containing GEM (50 mg/kg), SH2 TrM-(Arg)9 (5 mg/kg), or SH2 TrM-(Arg)9 (5 mg/kg) +GEM (20 mg/kg). B , C The tumor volumes of PANC-1 ( B ) and PANC-1/GEM ( C ) mouse models in different groups are shown ( n = 5). D Tumor weights of PANC-1 and PANC-1/GEM mice in different groups. ( n = 5). E , F Representative histological examination of the dissected tumors using HE staining, TUNEL assay, and Ki67 staining of PANC-1 ( E ) and PANC-1/GEM ( F ) mouse models. G Representative histological examination of the dissected tumors using LC3 and p62 staining. H Western blot of LC3 and p62 in tumors with different treatments. I Western blot of pY, pmTOR, mTOR, pEIF4EBP1, EIF4EBP1, pDDR1, DDR1, pPYK2, PYK2, pERK1/2, and ERK1/2 in tumors with different treatments. ( n = 5). Results are representative of three independent experiments with similar results. ** P < 0.01, *** P < 0.001.

Article Snippet: H , I Western blot of pDDR1, DDR1, pPYK2, PYK2, pERK1/2, ERK1/2, LC3, and p62 after treatment with collagen or PF431396 (T2314, Targetmol, USA) and Selumetinib (T6218, Targetmol, USA).

Techniques: Injection, Staining, TUNEL Assay, Western Blot