Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Primers sequences used for polymerase chain reaction (PCR).

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Polymerase Chain Reaction

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) was highly expressed in non‐small cell lung cancer (NSCLC) tissues and cells. (a) USP22 expression in NSCLC tissues was evaluated by immunohistochemistry (IHC) assay. (b, c) USP22 mRNA and protein levels in NSCLC tissues and adjacent normal tissues were measured by qRT‐PCR and western blot assays. (d, e) The mRNA and protein levels of USP22 in 16HBE, PC9, and HCC827 cells were determined by qRT‐PCR assay and western blot assay, respectively. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Deficiency of ubiquitin‐specific peptidase 22 (USP22) repressed non‐small cell lung cancer (NSCLC) cell migration and aggravated apoptosis and ferroptosis. (a) The protein level of USP22 in sh‐NC or sh‐USP22‐transfected PC9 and HCC827 cells was measured by western blot. (b) The migration of PC9 and HCC827 cells with sh‐NC or sh‐USP22 transfection was evaluated by transwell assay. (c) The apoptosis of PC9 and HCC827 cells transfected with sh‐NC or sh‐USP22 was analyzed by flow cytometry analysis. (d–g) The levels of reactive oxygen species (ROS), MDA, Fe 2+ , and GSH in sh‐NC or sh‐USP22 transfected PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Migration, Transfection, Western Blot, Transwell Assay, Flow Cytometry

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) overexpression promoted gefitinib resistance and repressed ferroptosis in gefitinib‐treated non‐small cell lung cancer (NSCLC) cells. (a) USP22 protein level in gefitinib or dimethyl sulfoxide (DMSO)‐treated PC9 and HCC827 cells was measured by western blot. (b) The effect of USP22 on gefitinib resistance was assessed by CCK‐8 assay. (c–i) PC9 and HCC827 cells were treated with DMSO+pcDNA, gefitinib+pcDNA, or gefitinib+USP22. (c, d) The migration and apoptosis of PC9 and HCC827 cells were evaluated by transwell assay and flow cytometry analysis, respectively. (e, f) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (g–i) The levels of MDA, Fe 2+ , and glutathione (GSH )in PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Over Expression, Western Blot, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) stabilized murine double minute 2 (MDM2) and modulated MDM2 expression. (a) Ubibrowser software predicted the relation between USP22 and MDM2. (b) Pull‐down assay was conducted to analyze the level of MDM2 ubiquitination. (c) Co‐IP assay was performed to explore the interaction between USP22 and MDM2. (d) The expression of MDM2 in non‐small cell lung cancer (NSCLC) tissues and normal tissues was estimated through IHC assay. (e, f) The mRNA and protein levels of MDM2 in NSCLC tissues and normal tissues were measured by qRT‐PCR and western blot, respectively. (g, h) The mRNA and protein levels of MDM2 in 16HBE, PC9, and HCC827 cells were examined by qRT‐PCR and western blot, respectively. (i) After PC9 and HCC827 cells were treated with DMSO, gefitinib, gefitinib+USP22, or gefitinib+USP22 + sh‐MDM2, MDM2 protein level was measured by western blot. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Expressing, Software, Pull Down Assay, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Murine double minute 2 (MDM2) knockdown reversed the effects of Ubiquitin‐specific peptidase 22 (USP22) overexpression on gefitinib sensitivity and ferroptosis in gefitinib‐treated NSCLC cells. (a) IC50 of gefitinib in PC9 and HCC827 cells transfected with pcDNA, USP22, or USP22 + sh‐MDM2 was estimated through CCK‐8 assay. (b–h) PC9 and HCC827 cells were treated with dimethyl sulfoxide (DMSO), gefitinib, gefitinib+USP22, gefitinib+USP22 + sh‐NC, or gefitinib+USP22 + sh‐MDM2. (b, c) The migration and apoptosis of PC9 and HCC827 cells were explored by transwell assay and flow cytometry analysis. (d, e) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (f–h) The levels of MDA, Fe 2+ , and glutathione (GSH) in PC9 and HCC827 cells were determined with commercial kits. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Knockdown, Ubiquitin Proteomics, Over Expression, Transfection, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) enhanced gefitinib resistance of non‐small cell lung cancer (NSCLC) cells in vivo. (a) The protein levels of USP22 and murine double minute 2 (MDM2) in PBS, gefitinib, and gefitinib+USP22 treated PC9 cells were measured by western blot. (b) Xenograft tumor volume was monitored every 5 days. (c) Xenograft tumor weight was examined after 25 days. (d) The expression of USP22 and MDM2 in xenograft tumors was examined through immunohistochemistry (IHC) assay. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, In Vivo, Western Blot, Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: The Immune System Strikes Back: Cellular Immune Responses against Indoleamine 2,3-dioxygenase

doi: 10.1371/journal.pone.0006910

Figure Lengend Snippet: ( a ), Lysis by the IDO5-specific T-cell clone (RBS35) of the HLA-A2 + breast cancer cell lines CAMA-1 (right) and MDA-MB-231 (left) before and after IFN-γ treatment. ( b ) , Histograms showing intracellular IDO expression in CAMA-1 before and after IFN-γ treatment. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI IDO (dark histograms) and MFI Isotype control (light histograms), where MFI is the Mean Fluorescence Intensity. The fold of expression was defined as MFI IDO /MFI Isotype control . Top: CAMA-1 ( p = 0.020 and MFI IDO /MFI Isotype control = 2.3). Bottom: CAMA-1 + IFN-γ treatment ( p = 0.004 and MFI IDO /MFI Isotype control = 3.5). ( c ), Histograms showing HLA-A2 expression in CAMA-1 before and after IFN-γ treatment. Data are representative of 3 experiments. HLA-A2 expression was given by a one-tailed two sampled t-test comparing MFI HLA-A2 (dark histograms) and MFI Isotype control (light histograms). The fold of expression was defined as MFI HLA-A2 /MFI Isotype control . Top: CAMA-1 ( p = 0.004 and MFI HLA-A2 /MFI Isotype control = 43.7). Bottom: CAMA-1 + IFN-γ treatment ( p = 0.002 and MFI IDO /MFI HLA-A2 = 141.2). ( d ) , Lysis of the colon cancer cell line SW480 transfected with IDO ShRNA for down-regulation of IDO protein expression by an IDO5-specific T-cell bulk culture. As a positive control, SW480 cells transfected with control ShRNA were used as target cells. All assays were performed in different E∶T ratios. ( e ) , Histograms showing intracellular IDO expression in SW480 transfected with control ShRNA ( p = 0.001 and MFI IDO /MFI Isotype control = 4.8) (top) and IDO ShRNA ( p = 0.040 and MFI IDO /MFI Isotype control = 2.1) (bottom) .

Article Snippet: Human SW480 cancer cells were transfected with indicated short hairpin RNA (ShRNA) plasmids obtained from SuperArray using FuGene6 (Roche) according to manufacturers instructions.

Techniques: Lysis, Expressing, One-tailed Test, Control, Fluorescence, Transfection, shRNA, Positive Control

Journal: Journal of Cancer

Article Title: Overexpression and biological function of PRDX6 in human cervical cancer

doi: 10.7150/jca.39892

Figure Lengend Snippet: The transduction efficiency of PRDX6 after transfecting SiHa cells for 48h. (A) Image of SiHa cells at normal light (× 100). (B) Image of SiHa cells infected with Lentiviruses vector pLVX-IRES-ZsGreen 1 at fluorescent. (C and D) The expression level of PRDX6 protein in SiHa cells transduced with PRDX6 overexpression vector. (E and F) PRDX6 protein expression in SiHa cells transfected with PRDX6 shRNA. Additionally, the names as control or WT, vector, PRDX6, Sh-Ctrl and Sh-PRDX6 represent blank control, an empty vector control of PRDX6 overexpression, overexpression of PRDX6, an empty vector control of PRDX6 knockdown and PRDX6 knockdown group, respectively. Data are expressed as mean ± standard deviation from triplicated experiments. ** P < 0.01.

Article Snippet: First, GFP-PRDX6 cDNA and non-silenced short hairpin RNA (shRNA) were generated in vitro by an outside company (Synbio Technologies, Shanghai, China).

Techniques: Transduction, Infection, Plasmid Preparation, Expressing, Over Expression, Transfection, shRNA, Control, Knockdown, Standard Deviation

Journal: Journal of Cancer

Article Title: Overexpression and biological function of PRDX6 in human cervical cancer

doi: 10.7150/jca.39892

Figure Lengend Snippet: The effect of PRDX6 on cervical cancer cell apoptosis. (A) Apoptosis percentage in SiHa cells with PRDX6 overexpression was evaluated by Annexin V-APC/7-AAD staining. Control: cDAN vetor control. (B) Apoptosis rate in PRDX6 knockdown group was analyzed by Annexin V-APC/7-AAD staining. Control: shRNA vetor control. (C and D) The expression levels of BAX and Bcl-2 in SiHa cells with upregulation or downregulation PRDX6 were measured by Western blot method. * P < 0.05, ** P < 0.01. Each bar represents mean ± standard deviation from triplicate experiments.

Article Snippet: First, GFP-PRDX6 cDNA and non-silenced short hairpin RNA (shRNA) were generated in vitro by an outside company (Synbio Technologies, Shanghai, China).

Techniques: Over Expression, Staining, Control, Knockdown, shRNA, Expressing, Western Blot, Standard Deviation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes

doi: 10.1152/ajpcell.00014.2011

Figure Lengend Snippet: Changes in the planar surface area of MCs in response to ANG II. A and B: representative photomicrographs of MCs used in contraction assays. A: MCs were cultured in NG (5.6 mM d-glucose + 20 mM l-glucose) and were transfected with either enhanced green fluorescent protein (EGFP) alone or EGFP plus shRNA construct specific for human TRPC6 (EGFP + hTRPC6-shRNA). B: MCs were cultured in HG (25.6 mM d-glucose) and were transfected with either EGFP alone or EGFP tagged TRPC6 expression plasmid (TRPC6-EGFP). The green cells indicate the positively transfected MCs and were used for analysis indicated by arrows. C: summary data from the experiments presented in A (Ca) and B (Cb) showing the contractile response of MCs with different treatments, calculated as [(the surface area of MCs after ANG II − the surface area of MCs before ANG II)/the surface area of MCs before ANG II] × 100%. n indicates the number of cells analyzed in each group. *P < 0.05; **P < 0.01 compared with EGFP group.

Article Snippet: As described by Du et al. ( 7 ) with modifications, human MCs were plated on a 22 × 22 mm coverslip and were transiently transfected with either green fluorescent protein (GFP) + hTRPC6-short hairpin RNA (shRNA) constructs (1:10, GFP:hTRPC6-shRNA) or TRPC6-enhanced GFP (EGFP) constructs using GenJet (SignaGen, Gaithersburg, MD).

Techniques: Cell Culture, Transfection, shRNA, Construct, Expressing, Plasmid Preparation