Journal: Frontiers in Endocrinology

Article Title: Identification of immune-related endoplasmic reticulum stress genes in proliferative diabetic retinopathy using bioinformatics analysis

doi: 10.3389/fendo.2024.1341206

Figure Lengend Snippet: External validation of TRAM1, TXNIP and ER stress-related hub genes (A) Validation of ER stress-related hub genes in the GSE60436 dataset. (B) The protein levels of TRAM1 and TXNIP were evaluated in cell samples by western blot. (C) The mRNA levels of BCL2, CCL2, IL-1Β, TLR4, TNF, and TP53 were measured in cell samples by qRT-PCR. Ctrl, control group; HG, high-glucose group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The membranes were then blocked in QuickBlockTM Western Blocking Buffer (P0252, Beyotime) for 30 minutes and incubated overnight at 4°C with primary antibodies against TRAM1 (18243-1-AP, Proteintech) and TXNIP (12705-1-AP, Proteintech).

Techniques: Biomarker Discovery, Western Blot, Quantitative RT-PCR, Control

Journal: Frontiers in Endocrinology

Article Title: Identification of immune-related endoplasmic reticulum stress genes in proliferative diabetic retinopathy using bioinformatics analysis

doi: 10.3389/fendo.2024.1341206

Figure Lengend Snippet: External validation of TRAM1, TXNIP and ER stress-related hub genes (A) Validation of ER stress-related hub genes in the GSE60436 dataset. (B) The protein levels of TRAM1 and TXNIP were evaluated in cell samples by western blot. (C) The mRNA levels of BCL2, CCL2, IL-1Β, TLR4, TNF, and TP53 were measured in cell samples by qRT-PCR. Ctrl, control group; HG, high-glucose group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The membranes were then blocked in QuickBlockTM Western Blocking Buffer (P0252, Beyotime) for 30 minutes and incubated overnight at 4°C with primary antibodies against TRAM1 (18243-1-AP, Proteintech) and TXNIP (12705-1-AP, Proteintech).

Techniques: Biomarker Discovery, Western Blot, Quantitative RT-PCR, Control

Journal: Regenerative Therapy

Article Title: The role of SFRP1 in human dermal papilla cell growth and its potential molecular mechanisms as a target in regenerative therapy

doi: 10.1016/j.reth.2024.12.001

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: Membranes were soaked in QuickBlockTM Western blocking buffer (P0252, Beyotime) for 50 min, then washed three times with a TBS-T buffer, and incubated overnight at 4 °C with primary antibodies against β-actin (Servicebio), SFRP1 (Abcam), β-catenin (Cell Signaling Technology) and TERT (Abcam).

Techniques:

Journal: Regenerative Therapy

Article Title: The role of SFRP1 in human dermal papilla cell growth and its potential molecular mechanisms as a target in regenerative therapy

doi: 10.1016/j.reth.2024.12.001

Figure Lengend Snippet: Expression levels of β-catenin and TERT mRNA and protein after transfection of SFRP1-specific DNA plasmids and siRNAs compared with the corresponding negative control. (A) mRNA level of β-catenin after pcDNA3.1-SFRP1 transfection; (B) mRNA level of TERT after pcDNA3.1-SFRP1 transfection; (C) mRNA level of β-catenin after siRNA-SFRP1 transfection; (D) mRNA level of TERT after siRNA-SFRP1 transfection; (E, F, G) protein levels of β-catenin and TERT after pcDNA3.1-SFRP1 transfection; (H, I, J) protein levels of β-catenin and TERT after siRNA-SFRP1 transfection. ∗ P ＜0.05, ∗∗ P ＜0.01, ∗∗∗ P ＜0.001, ∗∗∗∗ P ＜0.0001.

Article Snippet: Membranes were soaked in QuickBlockTM Western blocking buffer (P0252, Beyotime) for 50 min, then washed three times with a TBS-T buffer, and incubated overnight at 4 °C with primary antibodies against β-actin (Servicebio), SFRP1 (Abcam), β-catenin (Cell Signaling Technology) and TERT (Abcam).

Techniques: Expressing, Transfection, Negative Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin inhibits human non-small cell lung cancer by regulating AMPK–CEBPB–PDL1 signaling pathway

doi: 10.1007/s00262-021-03116-x

Figure Lengend Snippet: Metformin inhibited the proliferation of NSCLC. A Metformin has a concentration-dependent inhibitory effect on NSCLC cells. B, C The volcano map and heatmap show the differential expressed genes. D qPCR showed the mRNA of PDL1 in A549 and H1299 significantly reduced after metformin treatment. E The DNA fragment pulled by the CEBPB antibody was the fragment of − 95 to + 55 bp. F The PCR products were used for electrophoresis. Metformin concentration. NC: no metformin treatment. M1: 0.15 mmol/L (A549), 1 mmol/L (H1299). M2: 0.25 mmol/L (A549), 2 mmol/L (H1299)

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) and incubated with the following primary antibodies: CEBPB (Ab53138, Abcam), p-CEBPB (phosphor T235 + T188) (Ab52194, Abcam), AMPKα1 (Ab3759, Abcam), p-AMPKα1 (phosphor S48) ( Ab131357 , Abcam), PDL1 (13,684, Cell Signaling Technology), and actin (AA128, Beyotime Biotechnology).

Techniques: Concentration Assay, Electrophoresis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin inhibits human non-small cell lung cancer by regulating AMPK–CEBPB–PDL1 signaling pathway

doi: 10.1007/s00262-021-03116-x

Figure Lengend Snippet: Overexpression of LAP and LIP had opposite effects on PDL1 expression. A qPCR showed that the expression of CEBPB-mRNA was increased significantly in both A549 and H1299. B Western blot analysis showed that both the expression of CEBPB-LAP and CEBPB-LIP increased. The ratio of LIP/LAP increased. C The phosphorylated protein profile of A549 treated with metformin was analyzed. D The expression of pLAP*, pLAP, and pLIP phosphorylated levels in A549 cells was upregulated, but the ratio of pLIP/pLAP did not change significantly (0.849 vs. 0.849 vs. 0.850; P = 0.998). E The phosphorylated protein profile of H1299 treated with metformin was analyzed. F The expression of pLAP*, pLAP, and pLIP phosphorylated levels in H1299 cells was upregulated, but the ratio of pLIP/pLAP did not change significantly (3.282 vs. 3.458 vs. 3.443; P = 0.589). G CEBPB-LAP*, CEBPB-LAP, and CEBPB-LIP and control cell line CEBPB-NC cell lines were constructed. (H, I) Gene-chip results showed the differential expressed genes in A549-LAP-OE cells and LIP-OE cells. h Dual-luciferase reporter assays showed the RLU1/RLU2 of the LAP-OE group was significantly increased (7.146 vs. 8.128, P = 0.023), and the RLU1/RLU2 of the LIP-OE group was significantly reduced (7.146 vs. 5.830, P = 0.006). Metformin concentration. NC: no metformin treatment. M1: 0.15 mmol/L (A549), 1 mmol/L (H1299). M2: 0.25 mmol/L (A549), 2 mmol/L (H1299)

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) and incubated with the following primary antibodies: CEBPB (Ab53138, Abcam), p-CEBPB (phosphor T235 + T188) (Ab52194, Abcam), AMPKα1 (Ab3759, Abcam), p-AMPKα1 (phosphor S48) ( Ab131357 , Abcam), PDL1 (13,684, Cell Signaling Technology), and actin (AA128, Beyotime Biotechnology).

Techniques: Over Expression, Expressing, Western Blot, Control, Construct, Luciferase, Concentration Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin inhibits human non-small cell lung cancer by regulating AMPK–CEBPB–PDL1 signaling pathway

doi: 10.1007/s00262-021-03116-x

Figure Lengend Snippet: Metformin could inhibit the tumor growth in vivo. A, B, C The tumor growth of groups treated with metformin was significantly slower than that of groups not treated with metformin in nude mice. d, e Metformin could upregulate the CEBPB expression but downregulate the PDL1 expression in tumor tissues. f–h Metformin and atezolizumab could inhibit the tumor growth in C57BL/6 J mice, respectively, and atezolizumab and metformin has synergistic effect. i The immunohistochemistry staining showed that the expression PDL1 decreased in tumor of metformin group and atezolizumab group and decreased most significantly compared with those of control group

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) and incubated with the following primary antibodies: CEBPB (Ab53138, Abcam), p-CEBPB (phosphor T235 + T188) (Ab52194, Abcam), AMPKα1 (Ab3759, Abcam), p-AMPKα1 (phosphor S48) ( Ab131357 , Abcam), PDL1 (13,684, Cell Signaling Technology), and actin (AA128, Beyotime Biotechnology).

Techniques: In Vivo, Expressing, Immunohistochemistry, Staining, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin inhibits human non-small cell lung cancer by regulating AMPK–CEBPB–PDL1 signaling pathway

doi: 10.1007/s00262-021-03116-x

Figure Lengend Snippet: Metformin inhibited NSCLC through AMPK–CEBPB–PDL1 signaling pathway. A The proliferation of LAP-OE cells increased significantly (P < 0.05), and the proliferation of LIP-OE decreased significantly (P < 0.001) compared with NC cells. b, C The mobility of LAP-OE was significantly increased, while the mobility of LIP-OE was significantly decreased. D, E The cell migration of LAP-OE significantly increased, while the cell migration of LIP-OE significantly reduced. F No significant change in AMPKα1 mRNA expression was observed after being treated by metformin by qPCR. G, H The phosphorylation level of AMPKα1 (pAMPKα1) was significantly increased (P < 0.001), and the PDL1 protein was significantly downregulated in 549 cells and H1299 cells treated with metformin (P < 0.001). I, J After the addition of AMPK inhibitor (Compound C and Doxorubicin) in A549 cells and H1299 cells, AMPKα1 phosphorylation was significantly downregulated, and the expression of three isoforms of CEBPB, namely LAP*, LAP, and LIP, were all downregulated, while the expression of PDL1 was upregulated. The metformin reversed the inhibitory effect of AMPK inhibitors. K, l Western blot analysis showed that PDL1 expression was significantly increased LAP-OE cells, but was significantly downregulated in LIP-OE cells (P < 0.001). Metformin concentration. NC: no metformin treatment. M1: 0.15 mmol/L (A549), 1 mmol/L (H1299). M2: 0.25 mmol/L (A549), 2 mmol/L (H1299)

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) and incubated with the following primary antibodies: CEBPB (Ab53138, Abcam), p-CEBPB (phosphor T235 + T188) (Ab52194, Abcam), AMPKα1 (Ab3759, Abcam), p-AMPKα1 (phosphor S48) ( Ab131357 , Abcam), PDL1 (13,684, Cell Signaling Technology), and actin (AA128, Beyotime Biotechnology).

Techniques: Migration, Expressing, Phospho-proteomics, Western Blot, Concentration Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin inhibits human non-small cell lung cancer by regulating AMPK–CEBPB–PDL1 signaling pathway

doi: 10.1007/s00262-021-03116-x

Figure Lengend Snippet: Clinical analysis. A, B RNA transcriptome sequencing was performed on cancer tissue samples and identified the DEGs. C, D GO and KEGG analyses were performed. e, F Immunohistochemistry results showed that the expression of CEBPB in the metformin group was higher than that in the control group, and the expression of PDL1 was lower than that in the control group. G The survival analysis showed that patients with low expression of CEBPB-LAP had significantly better prognosis than those with high expression of CEBPB-LAP (HR = 1.19, 95% CI = 1.05–1.35, P = 0.0059). (H) Better prognosis was observed in patients with low PDL1 expression than high PDL1 expression (HR = 1.21, 95% CI = 1.02–1.42, P = 0.025)

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) and incubated with the following primary antibodies: CEBPB (Ab53138, Abcam), p-CEBPB (phosphor T235 + T188) (Ab52194, Abcam), AMPKα1 (Ab3759, Abcam), p-AMPKα1 (phosphor S48) ( Ab131357 , Abcam), PDL1 (13,684, Cell Signaling Technology), and actin (AA128, Beyotime Biotechnology).

Techniques: Sequencing, Immunohistochemistry, Expressing, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Metformin inhibits human non-small cell lung cancer by regulating AMPK–CEBPB–PDL1 signaling pathway

doi: 10.1007/s00262-021-03116-x

Figure Lengend Snippet: The clinical and pathological characteristics

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) and incubated with the following primary antibodies: CEBPB (Ab53138, Abcam), p-CEBPB (phosphor T235 + T188) (Ab52194, Abcam), AMPKα1 (Ab3759, Abcam), p-AMPKα1 (phosphor S48) ( Ab131357 , Abcam), PDL1 (13,684, Cell Signaling Technology), and actin (AA128, Beyotime Biotechnology).

Techniques: