Journal: Translational Cancer Research

Article Title: Sini decoction-polysaccharide compound regulates proliferation, apoptosis, and glycolysis of liver cancer cells through PHLDA2/ANXA2

doi: 10.21037/tcr-24-1625

Figure Lengend Snippet: Identification of difference-related genes affected by SNDPC in HCC. (A) Heatmaps, (B) volcano plot, (C,D) GO, (E,F) pathway, and (G,H) Venn were used to analyze the difference-related genes in HCC. (I) Western blotting was used to determine the expression of PHLDA2 and SPINK1. The results are presented as the mean ± SD. ***, P<0.001. GO, Gene Ontology; TCGA, The Cancer Genome Atlas; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SNDPC, Sini decoction-polysaccharide compound; HCC, hepatocellular carcinoma; SD, standard deviation.

Article Snippet: After the membranes were incubated overnight, they were sealed with 5% bovine serum albumin for 2 h and with primary antibodies against Bcl-2 (ab196495; Abcam, Cambridge, UK), Bax (ab32503; Abcam), cleaved caspase 3 (25128-1-AP; Proteintech, Rosemont, IL, USA), caspase 3 (82202-1-RR; Proteintech), LDHA [#2012; Cell Signaling Technology (CST), Danvers, MA, USA], HK2 (22029-1-AP; Proteintech), PKM2 (#3198; CST), PHLDA2 (14661-1-AP; Proteintech), SPINK1 (13477-1-AP; Proteintech), ANXA2 (#8235; CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118; CST) at 4 °C.

Techniques: Western Blot, Expressing, Standard Deviation

Journal: Translational Cancer Research

Article Title: Sini decoction-polysaccharide compound regulates proliferation, apoptosis, and glycolysis of liver cancer cells through PHLDA2/ANXA2

doi: 10.21037/tcr-24-1625

Figure Lengend Snippet: SNDPC regulated the expression of ANXA2 via PHLDA2. The (A) MEM database, (B) HCCDB, (C) LinkedOmics database, and (D) GEPIA database were used to determine the correlation between PHLDA2 and ANXA2. (E) ANXA2 protein level after treatment with 10–40 mg/mL if SNDPC. (F) A co-IP assay was used to confirm the combination of PHLDA2 and ANXA2. (G) The protein concentration of PHLDA2 after PHLDA2 overexpression was detected using Western blotting. (H) The protein concentration of PHLDA2 in HCCLM3 cells with PHLDA2 overexpression and SNDPC. The results are presented as the mean ± SD. ***, P<0.001; ### , P<0.001. TPM, transcripts per million; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ov-NC, empty vector; Ov-PHLDA2, PHLDA2-specific pcDNA-overexpression vector; pcDNA, protruding clustered DNA; SNDPC, Sini decoction-polysaccharide compound; MEM, Multi-Experiment Matrix; GEPIA, Gene Expression Profiling Interactive Analysis; co-IP, coimmunoprecipitation; SD, standard deviation.

Article Snippet: After the membranes were incubated overnight, they were sealed with 5% bovine serum albumin for 2 h and with primary antibodies against Bcl-2 (ab196495; Abcam, Cambridge, UK), Bax (ab32503; Abcam), cleaved caspase 3 (25128-1-AP; Proteintech, Rosemont, IL, USA), caspase 3 (82202-1-RR; Proteintech), LDHA [#2012; Cell Signaling Technology (CST), Danvers, MA, USA], HK2 (22029-1-AP; Proteintech), PKM2 (#3198; CST), PHLDA2 (14661-1-AP; Proteintech), SPINK1 (13477-1-AP; Proteintech), ANXA2 (#8235; CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118; CST) at 4 °C.

Techniques: Expressing, Co-Immunoprecipitation Assay, Protein Concentration, Over Expression, Western Blot, Plasmid Preparation, Gene Expression, Standard Deviation

Journal: Translational Cancer Research

Article Title: Sini decoction-polysaccharide compound regulates proliferation, apoptosis, and glycolysis of liver cancer cells through PHLDA2/ANXA2

doi: 10.21037/tcr-24-1625

Figure Lengend Snippet: PHLDA2 deficiency suppressed HCCLM3 cell proliferation ability, reduced glycolysis, and facilitated cell apoptosis. (A) The mRNA expression and (B) protein level of PHLDA2 in HCCLM3 cells were determined using qRT-PCR and Western blotting. (C) CCK-8 assay was used to determine cell proliferation. (D,E) The level of cell apoptosis was determined using TUNEL assay. (F) Western blotting was used to estimate apoptosis-related protein levels. (G) Glucose uptake, (H) lactate concentration, and (I) ECAR were detected after PHLDA2 silencing. (J) The protein levels of LDHA, HK2, and PKM2 in HCCLM3 cells were determined using Western blotting. The results are presented as the mean ± SD. ***, P<0.001. siRNA-NC, corresponding control siRNA; siRNA, small interfering RNA; siRNA-PHLDA2-1/2, specific siRNA against PHLDA2; mRNA, messenger RNA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ECAR, extracellular acidification rate; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; SD, standard deviation.

Article Snippet: After the membranes were incubated overnight, they were sealed with 5% bovine serum albumin for 2 h and with primary antibodies against Bcl-2 (ab196495; Abcam, Cambridge, UK), Bax (ab32503; Abcam), cleaved caspase 3 (25128-1-AP; Proteintech, Rosemont, IL, USA), caspase 3 (82202-1-RR; Proteintech), LDHA [#2012; Cell Signaling Technology (CST), Danvers, MA, USA], HK2 (22029-1-AP; Proteintech), PKM2 (#3198; CST), PHLDA2 (14661-1-AP; Proteintech), SPINK1 (13477-1-AP; Proteintech), ANXA2 (#8235; CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118; CST) at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, TUNEL Assay, Concentration Assay, Control, Small Interfering RNA, Real-time Polymerase Chain Reaction, Cell Counting, Standard Deviation

Journal: Translational Cancer Research

Article Title: Sini decoction-polysaccharide compound regulates proliferation, apoptosis, and glycolysis of liver cancer cells through PHLDA2/ANXA2

doi: 10.21037/tcr-24-1625

Figure Lengend Snippet: SNDPC suppressed HCC cell proliferation and glycolysis and enhanced apoptosis via PHLDA2/ANXA2. (A) CCK-8 assay was used to assess cell proliferation ability. (B,C) The level of cell apoptosis was determined using TUNEL assay. (D) Western blotting was used to determine the apoptosis-related proteins. (E) Glucose uptake, (F) lactate concentration, (G) and the ECAR were detected after PHLDA2 overexpression. (H) The protein levels of LDHA, HK2, and PKM2 in HCCLM3 cells were measured using Western blotting. The results are presented as the mean ± SD. ***, P<0.001; ### , P<0.001. SNDPC, Sini decoction-polysaccharide compound; Ov-NC, empty vector; Ov-PHLDA2, PHLDA2-specific pcDNA-overexpression vector; pcDNA, protruding clustered DNA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; ECAR, extracellular acidification rate; SD, standard deviation.

Article Snippet: After the membranes were incubated overnight, they were sealed with 5% bovine serum albumin for 2 h and with primary antibodies against Bcl-2 (ab196495; Abcam, Cambridge, UK), Bax (ab32503; Abcam), cleaved caspase 3 (25128-1-AP; Proteintech, Rosemont, IL, USA), caspase 3 (82202-1-RR; Proteintech), LDHA [#2012; Cell Signaling Technology (CST), Danvers, MA, USA], HK2 (22029-1-AP; Proteintech), PKM2 (#3198; CST), PHLDA2 (14661-1-AP; Proteintech), SPINK1 (13477-1-AP; Proteintech), ANXA2 (#8235; CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118; CST) at 4 °C.

Techniques: CCK-8 Assay, TUNEL Assay, Western Blot, Concentration Assay, Over Expression, Plasmid Preparation, Cell Counting, Standard Deviation

Journal: Translational Cancer Research

Article Title: Sini decoction-polysaccharide compound regulates proliferation, apoptosis, and glycolysis of liver cancer cells through PHLDA2/ANXA2

doi: 10.21037/tcr-24-1625

Figure Lengend Snippet: Identification of difference-related genes affected by SNDPC in HCC. (A) Heatmaps, (B) volcano plot, (C,D) GO, (E,F) pathway, and (G,H) Venn were used to analyze the difference-related genes in HCC. (I) Western blotting was used to determine the expression of PHLDA2 and SPINK1. The results are presented as the mean ± SD. ***, P<0.001. GO, Gene Ontology; TCGA, The Cancer Genome Atlas; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SNDPC, Sini decoction-polysaccharide compound; HCC, hepatocellular carcinoma; SD, standard deviation.

Article Snippet: After the membranes were incubated overnight, they were sealed with 5% bovine serum albumin for 2 h and with primary antibodies against Bcl-2 (ab196495; Abcam, Cambridge, UK), Bax (ab32503; Abcam), cleaved caspase 3 (25128-1-AP; Proteintech, Rosemont, IL, USA), caspase 3 (82202-1-RR; Proteintech), LDHA [#2012; Cell Signaling Technology (CST), Danvers, MA, USA], HK2 (22029-1-AP; Proteintech), PKM2 (#3198; CST), PHLDA2 (14661-1-AP; Proteintech), SPINK1 (13477-1-AP; Proteintech), ANXA2 (#8235; CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118; CST) at 4 °C.

Techniques: Western Blot, Expressing, Standard Deviation