Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 2 Ginsenoside Rg3 induced immunogenic cell death in CRC cells. (A) IC50 of Rg3 for CT26 and HCT116 cells at 24 h. Data are presented as mean SD (n Z 3). (B) Apoptosis (%) in CT26 and HCT116 cells following treatment of Rg3 at 6, 12 and 24 h. Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to DMSO. (C) The activity of UPR signaling pathways following treatment of Rg3 ([c] Z 30 mmol/L) at 6, 12 and 24 h. The quantification was demonstrated in Fig. S1. (D) The characterization of ICD in CRC cells following treatment of Rg3 ([c] Z 30 mmol/L), including CRT exposure (6 h), ATP secretion (12 h) and HMGB1 release (12 h). Data are presented as mean SD (n Z 3). *P < 0.05 relative to DMSO; scale bar Z 20 mm. (E) The expression of CD11c and CD86 in DCs stimulated with the supernatant from Rg3 ([c] Z 30 mmol/L) -treated cells (24 h). Data are presented as mean SD (n Z 3). **P < 0.01 relative to DMSO. (F) The in vivo vaccination assay using BALB/C and nude mice. Data are presented as mean SD (n Z 4). ***P < 0.001.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Activity Assay, Protein-Protein interactions, Expressing, In Vivo

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 3 Quercetin caused reactive oxygen species in CRC cells. (A) IC50 of QTN for CT26 and HCT116 cells at 24 h. Data are presented as mean SD (n Z 3). (B) Apoptosis (%) in CT26 and HCT116 cells following treatment of QTN ([c] Z 80 mmol/L) at 24 h. Data are presented as mean SD (n Z 3). **P < 0.01 relative to DMSO. (C) The activity of Bcl-2/BAX/caspase 9/caspase 3 signaling pathways following treatment of QTN ([c] Z 80 mmol/L) at 6, 12 and 24 h. The quantification was demonstrated in Fig. S2. (D) The ROS level in CT26 and HCT116 cells following treatment of QTN ([c] Z 80 mmol/L) at 6, 12 and 24 h. Data are presented as mean SD (n Z 3). **P < 0.01 and ***P < 0.001 relative to DMSO. (E) Cell viability (%) of CT26 and HCT116 cells with or without NAC prior to treatment of QTN ([c] Z 80 mmol/L) (24 h). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to untreated control. (F) Apoptosis (%) in CT26 and HCT116 cells with or without NAC prior to treatment of QTN ([c] Z 80 mmol/L) (24 h). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to untreated control.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Activity Assay, Protein-Protein interactions, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 4 Synergistic effects of Rg3 and QTN in CT26 cells. (A) IC50 of drug combination at 24 h. Data are presented as mean SD (n Z 3). CI values at IC50 were shown in Fig. S3. (B) Apoptosis (%) caused by drug combination at 24 h. Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to DMSO. (C) The CRT exposure with or without NAC before treatment of drug combination (6 h). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to DMSO; scale bar Z 20 mm. (D) The ATP secretion with or without NAC before treatment of drug combination at 12 h. Data are presented as mean SD (n Z 3).*P < 0.05 and **P < 0.01, between NAC and No NAC. (E) The HMGB1 release with or without NAC before treatment of drug combination at 12 h. Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01, between NAC and No NAC. (F) The expression of CD11c and CD86 in DCs stimulated (24 h) by the supernatant from Rg3- treated cells with or without pretreatment of NAC. Data are presented as mean SD (n Z 3). *P < 0.05, **P < 0.01 and ***P < 0.001, relative to DMSO.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Expressing

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 8 Combination therapy of targeted co-formulation and Anti-PD-L1 for CRC. (A) Treatment schedule and IVIS images. (B) The CRC progression over a 35-day period. Data are presented as mean SD (n Z 5). *P < 0.05 and **P < 0.01; NS, no significance. (C) Animal survival (median survival: PBS ~38 days, Anti-PD-L1 ~40 days, targeted co-formulation ~62 days, and combination z 96 days). Data are presented as mean SD (n Z 5). **P < 0.01 and ***P < 0.001. (D) Immunofluorescent staining assay (green Z DNA fragments and blue Z nuclei) on Day 20 to assess apoptosis in the tumor (scale bar Z 50 mm). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01, relative to PBS. (E) Level of immune cells in the tumor on Day 20 was analyzed using flow cytometry (BD). Data are presented as mean SD (n Z 4). *P < 0.05 and **P < 0.01; NS, no significance. (F) The mRNA expression of cytokines and chemokines in the tumor on Day 20 was analyzed using real time RT-PCR. Data are presented as mean SD (n Z 4). *P < 0.05 and **P < 0.01; NS, no significance. (G) Orthotopic CRC mice treated with targeted co-formulation following the removal of CD4 þ or CD8þ T cells. Data are presented as mean SD (n Z 4). *P < 0.05 and **P < 0.01; NS, no significance.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Formulation, Staining, Cytometry, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Comprehensive analysis of lactylation-related gene sets and mitochondrial functions in gastric adenocarcinoma: implications for prognosis and therapeutic strategies

doi: 10.3389/fimmu.2024.1451725

Figure Lengend Snippet: (A) RT-qPCR detected the knock-down efficiency of PTMA in NCI-N87 and MKN45 cell lines. (B) Cell viability of the MKN45 cell line before and after PTMA knockdown was detected by CCK8. (C) The cell viability of the NCI-N87 cell line before and after PTMA knockdown was detected by CCK8. (D) The apoptosis level of the MKN45 cell line before and after PTMA knockdown was detected by flow cytometry. (E) Western blot analysis assessed the expression of apoptosis-related proteins before and after PTMA knockdown in the MKN45 cell line. “**” denotes statistical significance (“**” p < 0.01). Sample sizes are indicated within the plots. Statistical comparisons were made using the Analysis of Variance (ANOVA).

Article Snippet: Following the manufacturer’s instructions, a Cell Apoptosis Detection Kit with Annexin V-mCherry and SYTOX Green (Beyotime, Shanghai, China) was used to identify cell apoptosis.

Techniques: Quantitative RT-PCR, Knockdown, Flow Cytometry, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

doi: 10.1111/jcmm.13707

Figure Lengend Snippet: AFAP1‐AS1 enhances cisplatin resistance in laryngeal carcinoma cells. A, HEp‐2 was treated with 4 μmol L −1 cisplatin. Expression of AFAP1‐AS1 was analysed at various times (0, 6, 12, 18, 24, and 30 h) by qRT‐PCR. * P < .05, ** P < .01, compared with 0 h. B, AFAP1‐AS1 silenced HEp‐2 cells were cultured in 96‐well plates. Cell viability was analysed by CCK8 assay under treatment with various concentration of cisplatin (0, 2, 4, 8, 16 and 32 μmol L −1 ). ** P < .01, compared with control siRNA transfected cells. C, Apoptosis assays in AFAP1‐AS1 silenced HEp‐2 cells under 8 μmol L −1 cisplatin treatment. ** P < .01, compared with control siRNA transfected cells. D, Cisplatin‐resistant HEp‐2 cell lines (HEp‐2/R) were established. Cell viability assays were performed in HEp‐2 and HEp‐2/R cells under various concentrations of cisplatin treatment. * P < .05, ** P < .01, compared with HEp‐2 cells. E, Expression of AFAP1‐AS1 in HEp‐2 and HEp‐2/R cells was analysed by qRT‐PCR. ** P < .01, compared with HEp‐2 cells

Article Snippet: We conducted nuclear DAPI staining to access cell apoptosis using Cell Apoptosis DAPI Detection Kit (GenScript, Piscataway, NJ) according to manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, CCK-8 Assay, Concentration Assay, Control, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

doi: 10.1111/jcmm.13707

Figure Lengend Snippet: miR‐320a reduces stemness and cisplatin resistance in laryngeal carcinoma cells. A, HEp‐2 cell morphology of parental cells and stemness‐enriched cell spheres (left) and corresponding miR‐320a expression (right). ** P < .01, compared with parental cells. B, Expression of miR‐320a in miR‐320 overexpression HEp‐2 cells by qRT‐PCR. *** P < .001, compared with control miRNA transfected cells. C, Expression of stemness‐associated genes in miR‐320a overexpression HEp‐2 cells. Gene expression was analysed by qRT‐PCR. * P < .05, ** P < .01, compared with control miRNA transfected cells. D, Number of tumour spheres in miR‐320a overexpression HEp‐2 cells. ** P < .01 compared with control miRNA transfected cells. E, miR‐320a overexpression HEp‐2 cells were cultured in 96‐well plates. Cell viability was analysed using CCK8 assays under various concentrations of cisplatin (0, 2, 4, 8, 16 and 32 μmol L −1 ). ** P < .01, compared with control miRNA transfected cells. F, Apoptosis assay in miR‐320a overexpression HEp‐2 cells under 8 μmol L −1 cisplatin treatment. ** P < .01, compared with control miRNA transfected cells. G, Expression of miR‐320a in HEp‐2 and HEp‐2/R cells was analysed by qRT‐PCR. ** P < .01, compared with HEp‐2 cells

Article Snippet: We conducted nuclear DAPI staining to access cell apoptosis using Cell Apoptosis DAPI Detection Kit (GenScript, Piscataway, NJ) according to manufacturer's instructions.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Control, Transfection, Gene Expression, Cell Culture, Apoptosis Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

doi: 10.1111/jcmm.13707

Figure Lengend Snippet: AFAP1‐AS1 regulates laryngeal carcinoma cells through miR‐320a/ RBPJ. A, Expression of RBPJ mRNA in AFAP1‐AS1 silenced, miR‐320a inhibition (miR‐320a‐in) and AFAP1‐AS1 silenced plus miR‐320a inhibition HEp‐2 cells by qRT‐PCR. * P < .05, compared with control cells. B, Expression of RBPJ protein in AFAP1‐AS1 silenced, miR‐320a inhibition and AFAP1‐AS1 silenced plus miR‐320a inhibition HEp‐2 cells by Western blot. C, Expression of RBPJ mRNA in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells by qRT‐PCR. * P < .05, compared with control cells. D, Expression of RBPJ mRNA in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells by Western blot. E, Number of tumour spheres in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells. * P < .05 compared with control cells. F, Apoptosis assay in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells under 8 μmol L −1 cisplatin treatment. * P < .05, compared with control cells

Article Snippet: We conducted nuclear DAPI staining to access cell apoptosis using Cell Apoptosis DAPI Detection Kit (GenScript, Piscataway, NJ) according to manufacturer's instructions.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Control, Western Blot, Apoptosis Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: In vivo CRISPR screening reveals that NFS1 deficiency enhances the sensitivity of CRC cells to oxaliplatin (Oxa). a Diagram showing the strategy of the CRISPR-based screen in vivo ( n = 6). b Volcano plot illustrating the depleted or enriched genes in the oxaliplatin-treatment group compared with the control group based on the depletion or enrichment of sgRNAs. Each dot represents a gene whose knockout can enhance (blue) or reduce (red) the sensitivity of cells to oxaliplatin treatment. c Illustration of the top ten candidates depleted in the oxaliplatin-treatment group. The analyzed CRISPR screening data are provided in Supplementary Table . d Schematic illustration of Fe–S cluster biogenesis and the main enzymes involved in this process. e MTS analysis of the proliferation of HCT116 cells in which NFS1 or FDX2 is silenced. f Quantification of colony formation analysis reflecting the proliferation of control and NFS1 -knockdown HCT116 and DLD1 cells. g , h Cell viability of HCT116 and DLD1 cells treated with different concentrations of oxaliplatin for 48 h after NFS1 knockdown. i , j LDH analysis indicating the cytotoxicity of different concentrations of oxaliplatin for 48 h in HCT116 and DLD1 cells with NFS1 knockdown. k Live/dead viability/cytotoxicity assay showing the dead (red) and live (green) cells among control and NFS1 -knockdown HCT116 cells treated with 40 µM oxaliplatin for 24 h. Scale bar = 100 μm. l Quantification of the relative number of dead cells in ( k ). m , n Cell viability ( m ) and cytotoxicity ( n ) assessments of control and NFS1 -knockdown HCT116 cells treated or not treated with 40 µM oxaliplatin for 24 h in combination with the apoptosis inhibitor Z-VAD-FMK (VAD, 25 µM), the necroptosis inhibitor necrostatin (Nec, 20 µM), the ferroptosis inhibitor ferrostatin-1 (Fer, 10 µM), the pyroptosis inhibitors Ac-DMPD/DMLD-CMK (DMPD/DMLD, 20 µM) and disulfiram (dis, 1 µM) or the autophagy inhibitor 3-methyladenine (3-me, 10 µM). The data in ( e – j ) and ( l – n ) are representative of three independent experiments and presented as the mean ± SD. The P values in ( e – h ) were calculated by two-way ANOVA with Dunnett’s multiple comparisons test, those in ( l – n ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test, and those in ( i , j ) were calculated by two-tailed unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The DAB (SA-HRP) TUNEL Cell Apoptosis Detection Kit (G1507, Solarbio Life Science, Beijing, China) was used to perform the TUNEL assay.

Techniques: In Vivo, CRISPR, Control, Knock-Out, Knockdown, Cytotoxicity Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 deficiency synergizes with oxaliplatin treatment to induce PANoptosis. a Representative images showing YP1 + cells (green) that may undergo apoptosis or necroptosis and PI + cells (red) that may undergo apoptosis, necroptosis, pyroptosis, or ferroptosis in control and NFS1 -knockdown HCT116 cells treated with 40 µM oxaliplatin for 24 h. The bottom panel shows representative bright fields, and the red arrowheads indicate the large bubbles emerging from the plasma membrane. Scale bar = 100 μm. b Quantification of the YP1 + and PI + cells from ( a ). c , d Flow cytometry ( c ) and quantification analysis ( d ) with Annexin V/PI staining evaluating the percentages of live cells (Annexin V − /PI − ), early apoptotic cells (Annexin V + /PI − ) and late apoptotic cells (Annexin V + /PI + ) among the control and NFS1 -knockdown DLD1 cells treated with PBS or oxaliplatin (80 μM, 24 h). e The lipid ROS levels in control and NFS1 -knockdown HCT116 cells treated with oxaliplatin (40 μM, 24 h) were assessed by the BODIPY™ 581/591 C11 probe assay. f – i Western blotting analysis of caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, phosphorylated MLKL, total MLKL, phosphorylated RIP1, total RIP1, cleaved RIP1, GSDME, cleaved GSDME, GSDMD, cleaved GSDMD, TFRC, FTH1 expression in control and NFS1 -knockdown HCT116 ( f , g ) and DLD1 ( h , i ) cells treated with oxaliplatin (40 μM for HCT116 and 80 μM for DLD1, 24 h), GSDME and GSDMD are not expressed in DLD1 cells. Vinculin was included as a loading control. The data in ( b , d , e ) are representative of three independent experiments and presented as the mean ± SD. The P values in ( b , d ) were calculated by two-way ANOVA and those in ( e ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The DAB (SA-HRP) TUNEL Cell Apoptosis Detection Kit (G1507, Solarbio Life Science, Beijing, China) was used to perform the TUNEL assay.

Techniques: Control, Knockdown, Clinical Proteomics, Membrane, Flow Cytometry, Staining, Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 deficiency enhances the antitumor effect of oxaliplatin in vivo. a , b Statistical analysis of CDX tumor volumes ( a ) and weights ( b ) in nude mice after implantation of NFS1 -knockdown or control DLD1 cells, followed by i.p. injection of oxaliplatin (7.5 mg/kg) or PBS ( n = 5, Bliss synergy P value is shown). c , d Quantification of the proliferation index (Ki67 staining) ( c ) and apoptotic index (TUNEL staining) ( d ) of DLD1-based xenograft tumors. e Illustration of the methodology used to establish CRC PDX models. f Photographs of the excised tumors from PDX #1 (left) and PDX #2 (right) models after intratumoral injection of in vivo-optimized NFS1 inhibitor (si NFS1 ) or the control, followed by i.p. injection of oxaliplatin (7.5 mg/kg) or PBS (PDX #1, n = 5; PDX #2, n = 4) and comparison of the tumor sizes. g , h Statistical analysis of the tumor volumes ( g , Bliss synergy P values are shown) and weights ( h ) in nude mice from the PDX #1 (left) and PDX #2 (right) models. i Representative H&E and IHC staining images of NFS1, Ki67, and TUNEL in PDX #1-based paraffin-embedded subcutaneous tumor sections. Scale bar = 50 μm. j , k Quantification of the proliferation index (Ki67 staining) ( j ) and apoptotic index (TUNEL staining) ( k ) of the PDX #1 (left) and PDX #2 (right) models. The data in ( a – d , g , h , j , k ) (PDX #1) are representative of five independent experiments and those in ( g , h , j , k ) (PDX #2) are representative of four independent experiments. All the data are presented as mean ± SD. The P values in ( a , g ) were calculated by two-way ANOVA, and those in ( b – d , h , j , k ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The DAB (SA-HRP) TUNEL Cell Apoptosis Detection Kit (G1507, Solarbio Life Science, Beijing, China) was used to perform the TUNEL assay.

Techniques: In Vivo, Knockdown, Control, Injection, Staining, TUNEL Assay, Comparison, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: Oxidative stress is critical for NFS1 deficiency-induced PANoptosis under oxaliplatin treatment. a Gene Ontology (GO) enrichment analysis of genes that exhibited twofold upregulation under oxaliplatin treatment (40 μM, 24 h) in the NFS1-knockdown group compared with the control group. b ROS analysis of control and NFS1 -knockdown HCT116 and DLD1 cells under PBS or oxaliplatin treatment (40 μM/80 μM, 24 h). c Cell viability of HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC or 5 mM GSH for 24 h after NFS1 knockdown. d Lipid ROS analysis of HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC for 24 h after NFS1 knockdown. e Representative images showing YP1 + cells (green) which may undergo apoptosis or necroptosis and PI + cells (red) which may undergo apoptosis, necroptosis, pyroptosis, or ferroptosis among control and NFS1 -knockdown HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC (24 h) after NFS1 knockdown. The bottom panel shows representative bright fields and the red arrowheads indicate the large bubbles emerging from the plasma membrane. Scale bar = 100 μm. f ROS analysis of control and NFS1 -knockdown HCT116 cells under cisplatin (40 μM, 24 h) or H 2 O 2 (100 μM, 24 h) treatment. g , h Cell viability of HCT116 cells treated with 100 μM H 2 O 2 ( g ) and 40 μM cisplatin ( h ) combined with 5 mM NAC for 24 h after NFS1 knockdown. i Cell cytotoxicity assessments of control and NFS1 -knockdown HCT116 cells under cisplatin (40 μM, 24 h) or H 2 O 2 (100 μM, 24 h) treatment. j Western blotting analysis of caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, phosphorylated MLKL, total MLKL, GSDME, cleaved GSDME and TFRC expression in control and NFS1 -knockdown HCT116 cells after treatment with 40 μM oxaliplatin combined with 5 mM NAC (24 h). k – m Photograph showing the gross comparison ( k ), tumor volumes ( l ), and weights ( m ) of control and NFS1 -knockdown HCT116 CDX tumors in nude mice subjected to i.p. injection of oxaliplatin (7.5 mg/kg) and NAC (1 mg/ml) in the drinking water ( n = 5). Vinculin was included as a loading control. The data in ( b – d , f – i ) are representative of three independent experiments and those in ( l , m ) are representative of five independent experiments, and all are presented as the mean ± SD. The P values in ( b , l ) were calculated by two-way ANOVA, and those in ( c , d , f – i , m ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001

Article Snippet: The DAB (SA-HRP) TUNEL Cell Apoptosis Detection Kit (G1507, Solarbio Life Science, Beijing, China) was used to perform the TUNEL assay.

Techniques: Knockdown, Control, Clinical Proteomics, Membrane, Western Blot, Expressing, Comparison, Injection

Journal: World Journal of Gastroenterology

Article Title: Chaperonin-containing tailless complex polypeptide 1 subunit 6A negatively regulates autophagy and protects colorectal cancer cells from cisplatin-induced cytotoxicity

doi: 10.3748/wjg.v31.i18.105729

Figure Lengend Snippet: Chaperonin-containing tailless complex polypeptide 1 subunit 6A negatively modulates apoptosis and necroptosis in colorectal cancer. A: After treating the indicated cells with cisplatin (DDP) for 24 hours, cell lysates were prepared and subjected to immunoblotting; B and C: Following treatment of the cells with DDP for 24 hours, the apoptosis were determined by flow cytometry (Annexin V-positive), and the data were visualized in histograms; D: Following treatment with DDP for 24 hours, cell lysates were performed immunoblotting with indicated antibodies; E and F: Cells were exposed to the appropriate drugs for 24 hours (necrostatin-1 [Nec-1]: 10 μM, hereafter unless otherwise indicated), cells were lysed, and immunoprecipitated using the receptor-interacting protein kinase 3 (RIP3) antibody. The immunoprecipitates were resolved by electrophoresis and probed by immunoblotting with the indicated antibodies; G: Cell viability in knockout (KO)-Control (Ctrl) and KO-chaperonin-containing tailless complex polypeptide 1 subunit 6a (CCT6A) cells was analyzed following the indicated treatments for 24 hours. b P < 0.01 vs control. Bcl-xL: B-cell lymphoma-extra large; IgG: Immunoglobulin G (negative control antibody); MLKL: Mixed lineage kinase domain-like; Nec-1; Necrostatin-1; OE: Overexpression; PARP-1; Poly(ADP-ribose) polymerase 1; TH: Total homogenate.

Article Snippet: A Cell Apoptosis Detection Kit (KGA1102-50) was obtained from KeyGene Biotech (Wageningen, Netherlands).

Techniques: Western Blot, Flow Cytometry, Immunoprecipitation, Electrophoresis, Knock-Out, Control, Negative Control, Over Expression