Journal: Cancer Immunology Research

Article Title: Blocking WNT7A Enhances MHC-I Antigen Presentation and Enhances the Effectiveness of Immune Checkpoint Blockade Therapy

doi: 10.1158/2326-6066.cir-24-0484

Figure Lengend Snippet: Figure 3. WNT7A inhibition suppresses tumor growth and reshapes the tumor immune landscape. A, Relative MHC-I (H-2Kb/H-2Db) and B2M expression in tumor cells from EO771 tumors were determined by flow cytometry (n ¼ 5, t test). B, Flow cytometry analysis of IFN-γ+ CD8+ T cells and TNF-α+ CD8+ T cells in shNC or shWnt7a EO771 tumors (n ¼ 5, t test). C, shNC or shWnt7a EO771 tumor growth (n ¼ 5 mice/group, two-way ANOVA). D, Tumor growth (middle) and survival analysis (right) of EO771 tumor-bearing mice with indicated treatment. Mice were injected with anti-CD8 neutralizing antibodies or isotype control (IgG). Peripheral blood was collected to examine depletion of CD8. The percentage of CD8+ cells in CD45+ populations is shown (left). n ¼ 5 mice/group, two-way ANOVA for tumor volume comparison, log-rank test for survival comparison. E–H, EO771 cells transduced with WNT7A shRNA and/or B2M shRNA were orthotopically implanted into C57BL/6 mice (n ¼ 7 per group). Tumor growth (E); cell surface MHC-I and B2M in EO771 tumors of the indicated groups (F); quantification of CD8+ T cells (G); and IFNγ+ and TNFα+ CD8+ T cells in EO771 tumors of the indicated groups (H). I, tSNE plot of tumor-infiltrating leukocytes overlaid with color-coded clusters in shNC or shWnt7a EO771 tumors. J, Frequency of clusters of indicated immune cell subsets from shNC or shWnt7a EO771 tumors (n ¼ 5, t test). MFI, mean fluorescence intensity; tSNE, t-distributed stochastic neighbor embedding.

Article Snippet: The lentivirus was generated by transfecting HEK293T cells (RRID: CVCL_0063) with Lipofectamine 2000 (Invitrogen, 11668019) 36 μL, 6 μg of lentiviral shRNA plasmid, 1.5 μg of the viral packaging plasmid psPAX2 (RRID: Addgene 12260), and 4 μg of the envelope plasmid pMD2.G (RRID: Addgene 12259).

Techniques: Inhibition, Expressing, Flow Cytometry, Injection, Control, Comparison, Transduction, shRNA, Fluorescence

Journal: Journal of Cell Communication and Signaling

Article Title: TGF-β1 and TNF-α synergistically induce epithelial to mesenchymal transition of breast cancer cells by enhancing TAK1 activation

doi: 10.1007/s12079-019-00508-8

Figure Lengend Snippet: The up-regulation of TGF-β receptors contributes to the gradually enhanced activation of TAK1 during long-lasting co-stimulation. a–c MCF-7 cells were cultured in absence or presence of TGF-β1/TNF-α (left) for the indicated time. Or the cells were cultured for 6 days in presence of TGF-β1 and or TNF-α. The expression of TGF-βRI (a), and TGF-βRII (b) was detected by real-time RT-PCR. c The expression of TβRI and TβRII was detected by Western blot after 6-d culture (left). Relative expression of TβRI and TβRII were calculated after densitometry assay as standardized by β-actin (right). d MCF-7 cells were unstimulated or stimulated with TGF-β1/TNF-α in absence or presence of SIS3 (10 μM), QNZ (40 nM), SB203580 (SB, 10 μM), PD98059 (PD, 10 μM) and SP600125 (SP, 10 μM) for 6 days. The expression of TGF-βRI (left) and TGF-βRII (right) was detected by real-time RT-PCR. e–h MCF-7 cells were transducted with control, TβRI or TβRII shRNA lentivirus. And then the cells were selected for stable expression using puromycin. e The expression of TβRI and TβRII was detected by Western blot (left). Relative expression of TβRI and TβRII was calculated after densitometry assay as standardized by β-actin (right). f The phospho-TAK1, TAK1, phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by Western blot. g The activity of NF-κB was assayed as described in Methods. h The ratio of phosphorylated protein to total protein of p38 MAPK, ERK1/2 and JNK was calculated after densitometric analysis of the blots. Data are representative of three independent experiments, or pooled from three independent experiments. P values, * P < 0.05, **P < 0.01

Article Snippet: Cell transduction Control shRNA lentiviral particles (sc-108,080), TβRI lentiviral particles (sc-40,222-V), TβRII shRNA lentiviral particles (sc-36,658-V) and SLUG shRNA lentiviral particles (sc-38,393-V) were all purchased from Santa Cruz Biotechnology (sc-).

Techniques: Activation Assay, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Control, shRNA, Activity Assay

Journal: Journal of Cell Communication and Signaling

Article Title: TGF-β1 and TNF-α synergistically induce epithelial to mesenchymal transition of breast cancer cells by enhancing TAK1 activation

doi: 10.1007/s12079-019-00508-8

Figure Lengend Snippet: The sustained activation of signaling pathways and up-regulation of SLUG are required for inducing EMT and invasiveness in breast cancer cells. a and b MCF-7 cells were stimulated or unstimulated with TGF-β1 and TNF-α for 6 days in absence or presence of (5Z)-7-oxozeaenol. a The expression of E-cadherin and vimentin was detected by Western blot. b The cells were used for matrigel invasion assay. c and d MCF-7 cells were stimulated or unstimulated with TGF-β1 and TNF-α for 6 days in absence or presence of SIS3, QNZ, SB203580, PD98059 and SP600125 for 6 days. c The expression of E-cadherin and vimentin was detected by Western blot (left). Relative expression of E-cadherin and vimentin were calculated after densitometry assay as standardized by β-actin (right). d The cells were used for matrigel invasion assay. e MCF-7 cells were cultured in presence of TGF-β1 and/or TNF-α for 6 days. The expression of SLUG, SNAIL, ZEB1 and TWIST1 was detected by real-time RT-PCR. f–h MCF-7 cells were transducted with control or SLUG shRNA lentivirus, and then selected for stable expression using puromycin. f The expression of SLUG, E-cadherin, vimentin and β-actin was detected by Western blot. g Relative expression of SLUG, E-cadherin and vimentin were calculated after densitometry assay as standardized by β-actin. h The cells were used for matrigel invasion assay. Data are representative of three independent experiments, or pooled from three independent experiments. P values, * P < 0.05, **P < 0.01

Article Snippet: Cell transduction Control shRNA lentiviral particles (sc-108,080), TβRI lentiviral particles (sc-40,222-V), TβRII shRNA lentiviral particles (sc-36,658-V) and SLUG shRNA lentiviral particles (sc-38,393-V) were all purchased from Santa Cruz Biotechnology (sc-).

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Western Blot, Invasion Assay, Cell Culture, Quantitative RT-PCR, Control, shRNA

Journal: Endocrine Journal

Article Title: Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic β-cells

doi: 10.1507/endocrj.ej16-0338

Figure Lengend Snippet: Fig. 1 Expression of 293 GPCRs in human pancreatic islet (A) Mean expression level of 293 GPCRs performed by qPCR in isolated human islets (n=3-4 donors) illustrating that GPRC5C is one of the most abundantly expressed GPCRs in human islets. (B) Pie chart of functional annotations of the 388 human islet genes strongly associated with the mRNA expression of GPRC5C: 56.4% of the genes with known function (298 genes) regulate cell death and survival, 13.4% (71 genes) regulate cell cycle and 30.1% (159 genes) regulate carbohydrate and lipid metabolism. Please note that several genes were annotated to more than one cellular function category. The remaining 806 strongly associated genes have as yet unknown functions relating to β-cell function and diabetes. Data are based on 89 individual human islet preparations.

Article Snippet: Freshly isolated mouse islets were cultured in 1 ml RPMI 1670 medium (Statens Veterinärmedicinska Anstalt, Sweden) for 36 h in the presence of a pool of five different Gprc5c shRNA lentiviral particles or scrambled control lentiviral particles according to the manufacturer’s recommendations (Santa Cruz Biotechnology).

Techniques: Expressing, Isolation, Functional Assay, Cell Function Assay

Journal: Endocrine Journal

Article Title: Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic β-cells

doi: 10.1507/endocrj.ej16-0338

Figure Lengend Snippet: Fig. 2 CPRC5C expression in mouse and human pancreatic islets (A) Gprc5c expression in different mouse tissues analysed by western blotting. A representative image of Gprc5c protein expression in mouse brain, lung, heart, liver, kidney and pancreatic islets. For comparison, the protein expression of β-actin in each tissue is also shown (B) Confocal microscopy of mouse islets labelled for Gprc5c (green fluorescence) and insulin (red fluorescence) and the overlay of the two (yellow). Scale bar: 20 μm. (C) Expression of Gprc5a, Gprc5b, Gprc5c, Gprc5d and Glp1r mRNA relative to Gapdh mRNA analysed by qPCR in isolated mouse pancreatic islets (n=6 in each group). (D) Comparison of mRNA expression of GPRC5A, GPRC5B, GPRC5C, GPRC5D and GLP1R analysed by qPCR in human islets relative to GAPDH (n=3 donors). (E) mRNA expression of GPRC5C (n=15 in each group) normalised to three separate housekeeping genes (GAPDH, HPRT and PPAI) in human islets from non-diabetic (Non-Diab) and type-2 diabetic (Diab) islet donors. (F) Representative western blot of GPRC5C protein expression in human islets from non-diabetic and diabetic donors. Mean band intensities (means ± SEM) are presented. The blot for the endogenous control protein β-actin is also shown (n=4). (G) Gprc5c mRNA expression relative to Gapdh mRNA in newborn mouse islets plotted relative the mRNA expression of Gprc5c in adult mouse islets (n=5-6 in each group). (H) Fold change differences in intensity of western blot bands showing Gprc5c protein expression relative to β-actin in isolated islets of newborn (<3 weeks) and adult (>46 weeks) mice analysed by qPCR (n=10-16 in each group). * p<0.05, ** p<0.01.

Article Snippet: Freshly isolated mouse islets were cultured in 1 ml RPMI 1670 medium (Statens Veterinärmedicinska Anstalt, Sweden) for 36 h in the presence of a pool of five different Gprc5c shRNA lentiviral particles or scrambled control lentiviral particles according to the manufacturer’s recommendations (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Comparison, Confocal Microscopy, Fluorescence, Isolation, Control

Journal: Endocrine Journal

Article Title: Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic β-cells

doi: 10.1507/endocrj.ej16-0338

Figure Lengend Snippet: Fig. 3 CPRC5C activation and down stream signalling molecules (A) The effect of Gprc5c knock-down on isolated mouse islet cAMP content incubated at low (1 mmol/l) or high (16.7 mmol/l) glucose in the presence or absence of ATRA (5 μmol/l) for 60 min. (B) Addition of ATRA (10 µM) causes increased Ca2+ oscillations in Huh7 cells over-expressing the GPRC5C receptor as measured by FLIPR. (C) Signalling pathways associated with cell cycle progression (E2F), MAPK/ERK (SRE), Ca2+ mobilization (NFAT) and β-cell function (PAX6, HNF4) are more active in cells over-expressing the GPRC5C receptor, whereas the pro-inflammatory transcription factor NFκB is inhibited. GPRC5C over-expression did not affect STAT3 and PPAR transcription factor activity. Data presented as log2 fold change of the mean of transcription factor activity in GPRC5C over-expressing Huh7 cells vs. wt Huh7 cells. (D) Western blots showing phosphorylated ERK1/2 (pERK1/2) relative to GAPDH in wt or HUH7-cells transfected with GPRC5C and densitometry analysis of pERK1/2 bands relative to GAPDH. Mean ± SEM are presented for 4 observations in each group. (E) The proliferative effect on GPRC5C over-expressing Huh7 cells vs. wt were evaluated by BrdU incorporation. Dashed line denotes wt cell proliferation in the absence of FBS. Data are means ± SEM of 4 independent experiments in duplicate. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Freshly isolated mouse islets were cultured in 1 ml RPMI 1670 medium (Statens Veterinärmedicinska Anstalt, Sweden) for 36 h in the presence of a pool of five different Gprc5c shRNA lentiviral particles or scrambled control lentiviral particles according to the manufacturer’s recommendations (Santa Cruz Biotechnology).

Techniques: Activation Assay, Knockdown, Isolation, Incubation, Expressing, Cell Function Assay, Over Expression, Activity Assay, Western Blot, Transfection, BrdU Incorporation Assay

Journal: Discover. Oncology

Article Title: RPN1 promotes the proliferation and invasion of breast cancer cells by activating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1007/s12672-024-00875-8

Figure Lengend Snippet: Fig. 1 RPN1 mRNA expression level is increased in breast cancer tissue/cells. A qRT-PCR assay for RPN1 mRNA expression in 25 paired breast cancer tissues and adjacent non-tumor tissue samples. B qRT-PCR assay for RPN1 mRNA expression in breast cancer cell line MCF7 and nor- mal human breast epithelial cell line MCF10A. * * P < 0.05

Article Snippet: To generate RPN1 knockdown cell models, RPN1 shRNA lentiviral particles (sc-36,420-V, Santa Cruz) or control shRNA lentiviral particles (sc108060, Santa Cruz) were transfected into MCF7 cells according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: Discover. Oncology

Article Title: RPN1 promotes the proliferation and invasion of breast cancer cells by activating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1007/s12672-024-00875-8

Figure Lengend Snippet: Fig. 2 The effect of RPN1 knockdown on the PI3K/AKT/mTOR signaling axis. A qRT-PCR assay for the measurement of RPN1 mRNA expres- sion in shRNA RPN1 transfected cells. B Western blot for the measurement of RPN1 protein expression in shRNA RPN1 transfected cells. C Western blot for the measurement of the PI3K/AKT/mTOR and their phosphorylation expression. The ratios of p-PI3K/PI3K (D), p-AKT/AKT (E) and p-mTOR/mTOR (F) were statistically analyzed. ** P < 0.05 (vs. sh-NC group). ##&& P < 0.05 (vs. 740Y-P + sh-NC group)

Article Snippet: To generate RPN1 knockdown cell models, RPN1 shRNA lentiviral particles (sc-36,420-V, Santa Cruz) or control shRNA lentiviral particles (sc108060, Santa Cruz) were transfected into MCF7 cells according to the manufacturer’s instructions.

Techniques: Knockdown, Quantitative RT-PCR, shRNA, Transfection, Western Blot, Expressing, Phospho-proteomics

Journal: Discover. Oncology

Article Title: RPN1 promotes the proliferation and invasion of breast cancer cells by activating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1007/s12672-024-00875-8

Figure Lengend Snippet: Fig. 4 RPN1 promotes migration and invasion of breast cancer cells via the PI3K/AKT/mTOR signaling pathway. A Transwell assay in sh-NC or sh-RPN1 transfected MCF7 cells with or without treatment of 740Y-P, a PI3K activator. B Wound healing assay in sh-NC or sh-RPN1 trans- fected MCF7 cells with or without treatment of 740Y-P. **P < 0.05 (vs. sh-NC group), ##&& P > 0.05 (vs. 740Y-P + sh-NC group)

Article Snippet: To generate RPN1 knockdown cell models, RPN1 shRNA lentiviral particles (sc-36,420-V, Santa Cruz) or control shRNA lentiviral particles (sc108060, Santa Cruz) were transfected into MCF7 cells according to the manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Transfection, Wound Healing Assay

Journal: Discover. Oncology

Article Title: RPN1 promotes the proliferation and invasion of breast cancer cells by activating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1007/s12672-024-00875-8

Figure Lengend Snippet: Fig. 3 RPN1 promotes the proliferation of breast cancer cells via the PI3K/AKT/mTOR signaling pathway. A Cell availability assay in sh-NC or sh-RPN1 transfected MCF7 cells with or without treatment of 740Y-P. B Apoptosis analyzed by flow cytometry in sh-NC or sh-RPN1 trans- fected MCF7 cells with or without treatment of 740Y-P. **P < 0.05 (vs. sh-NC group), ##&& P > 0.05 (vs. 740Y-P + sh-NC group)

Article Snippet: To generate RPN1 knockdown cell models, RPN1 shRNA lentiviral particles (sc-36,420-V, Santa Cruz) or control shRNA lentiviral particles (sc108060, Santa Cruz) were transfected into MCF7 cells according to the manufacturer’s instructions.

Techniques: Transfection, Flow Cytometry

Journal: Frontiers in oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates.

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: FIGURE 1 CEACAM5 expressions in different normal/tumor tissues and DM4 sensitivity in NSCLCs and PDACs. (A) CEACAM5 immunohistochemistry (IHC) of 14 tumor tissues and 14 correspondent normal tissues in tissue microarray (left panel) and quantification values of IHC images (right panel). (B) DM4 response curves in NSCLCs (H1975, A549, H1299, H2030, and H2009 cells) and PDACs (HPAC and HPAF-II cells). Normalized % cell viability (ATP level) was calculated by normalizing luminescence values using buffer (DMSO)-treated respective cells. Results are shown as the mean ±SD for representative data from three independent experiments.

Article Snippet: H1975-CEACAM5, H2009-CEACAM5, A549-CEACAM5, and H1299-CEACAM5, stably expressing CEACAM5, were generated by stable infection with lentivirus from the CEACAM5 lentiviral plasmid (Origene) using a commonly used protocol (36).

Techniques: Immunohistochemistry, Microarray

Journal: Frontiers in oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates.

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: FIGURE 2 in vitro cell killing activities of the ADC SAR408701 analog and CAR-T cells targeting CEACAM5 in DM4S cells. (A) Cell surface CEACAM5 expression levels in DM4S NSCLCs (H1299, H1299-CEACAM5, A549, and A549-CEACAM5 cells) and PDACs (HPAC and HPAF-II cells). (B, D) Cell killing activities with ADC SAR408701 analog against DM4S NSCLCs (H1299, H1299-CEACAM5, and A549, A549-CEACAM5 cells) (B) and DM4S PDACs (HPAC, and HPAF-II cells) (D). Normalized % cell viability (ATP level) was calculated by normalizing luminescence values using vehicle (buffer)-treated respective cells. The IC50 were then determined by nonlinear regression plot of percent specific cytotoxicity versus Log10 concentration of ADC SAR408701 analog using GraphPad Prism software. (C, E) Cytotoxic activities (%) of anti-CEACAM5 CAR-T cells against DM4S NSCLCs (C) and PDACs (E). Significance was tested using one-way ANOVA, followed by the tukey’s multiple post hoc test. ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05; versus control T at each E:T ratio. (B–E) Results are shown as the mean ±SD for representative data from three independent experiments.

Article Snippet: H1975-CEACAM5, H2009-CEACAM5, A549-CEACAM5, and H1299-CEACAM5, stably expressing CEACAM5, were generated by stable infection with lentivirus from the CEACAM5 lentiviral plasmid (Origene) using a commonly used protocol (36).

Techniques: In Vitro, Expressing, Concentration Assay, Software, Control

Journal: Frontiers in oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates.

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: FIGURE 3 in vitro cell cytotoxicity assay of the ADC SAR408701 analog and CAR-T cells targeting CEACAM5 in DM4R cells. (A) Cell surface CEACAM5 expression levels in DM4R NSCLCs (H1975, H1975-CEACAM5, H2009, and H2009-CEACAM5 cells). (B) Cell killing activities with ADC SAR408701 analog against CEACAM5-positive DM4R cell lines (H1975-CEACAM5 and H2009-CEACAM5 cells) and CEACAM5-negative DM4R cell lines (H1975 and H2009 cells). Normalized % cell viability (ATP level) was calculated by normalizing luminescence values for vehicle (buffer)-treated respective cells. (C) Cytotoxic activities (%) of anti-CEACAM5 CAR-T cells against CEACAM5-positive and CEACAM5-negative DM4R NSCLC cells. Significance was tested using one-way ANOVA, followed by the tukey’s multiple post hoc test. ****, P<0.0001; ***, P<0.001; versus control T at each E:T ratio. (B, C) Results are shown as the mean ±SD for representative data from three independent experiments.

Article Snippet: H1975-CEACAM5, H2009-CEACAM5, A549-CEACAM5, and H1299-CEACAM5, stably expressing CEACAM5, were generated by stable infection with lentivirus from the CEACAM5 lentiviral plasmid (Origene) using a commonly used protocol (36).

Techniques: In Vitro, Cytotoxicity Assay, Expressing, Control

Journal: Frontiers in oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates.

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: FIGURE 4 in vivo anti-tumor activities of the ADC SAR408701 analog and CAR-T cells targeting CEACAM5 in DM4S and DM4R NSCLC tumors. (A) Schematic representation of experimental design and treatment schedule for mice studies. (B, D, F) Tumor growth curve (left panels) and individual mice curves (right panels) of DM4S A549-CEACAM5 (B), DM4R H1975-CEACAM5 (D), and CEACAM5-negative DM4S A549 (F) tumors. Significance was analyzed by comparing the tumor volume (mm3) ±SD at endpoint of 1.0 cm3 (B) or end day (D, F) and determined using one-way ANOVA, followed by the tukey’s multiple post hoc test. ****, P<0.0001; ***, P<0.001. (C, E, G) Survival curve showing the efficacy of the ADC SAR408701 analog and CAR-T cells in NSG mice. Tumor volume (mm3) are shown as mean ±SD for n=6 or 7 per group. Survival was presented by Kaplan-Meier plot of percentage of mice with tumor volume ≥1-1.5 cm3. Significance was determined by log-rank (Mantel-Cox) test. ***, P<0.001; **, P<0.01.

Article Snippet: H1975-CEACAM5, H2009-CEACAM5, A549-CEACAM5, and H1299-CEACAM5, stably expressing CEACAM5, were generated by stable infection with lentivirus from the CEACAM5 lentiviral plasmid (Origene) using a commonly used protocol (36).

Techniques: In Vivo

Journal: Oncotarget

Article Title: miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

doi: 10.18632/oncotarget.14489

Figure Lengend Snippet: Listed protein and mRNA expressions in stable RPEs (A) and (B) and RGCs (C) and (D) with miR-141 (two lines, “L1/L2”) or scramble miRNA control (“miR-C”) were examined by Western blot assay ( A and C ) and RT-qPCR assay ( B and D ), respectively. Nuclear Nrf2 expression was also shown ( A and C , lower panels). Protein expressions of Nrf2, HO1 and NQO1 ( vs . β-actin) were quantified ( A and C ). For each assay, n=5. Experiments in this figure were repeated four times to insure consistency of results. * p <0.05 vs . “Parental” cells ( A – D ).

Article Snippet: The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours.

Techniques: Control, Western Blot, Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

doi: 10.18632/oncotarget.14489

Figure Lengend Snippet: Expression of miRNA-141 (A) Keap1 mRNA (B) and listed proteins ( B ) in RPEs transfected with antagomiR-141 or non-sense control antagomiR (“antagomiR-C”) were shown. Cells were also subjected to UV radiation (30 mJ/cm 2 ) and cultured for applied time, and cell viability was tested by MTT assay (C) Cell apoptosis was tested by Histone DNA ELISA assay (D) . “Transfection” indicates transfection reagents only. Protein expressions of Keap1, Nrf2, HO1 and NQO1 ( vs . β-actin) were quantified ( A and C ). For each assay, n=5. Experiments in this figure were repeated three times to insure consistency of results. * p <0.05 vs . “antagomiR-C” cells.

Article Snippet: The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours.

Techniques: Expressing, Transfection, Control, Cell Culture, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

doi: 10.18632/oncotarget.14489

Figure Lengend Snippet: miRNA-141-expressing RPEs (“L1/2”) were infected with lentiviral Nrf2 shRNA (“shNrf2-A/-B”, with non-overlapping sequence) or scramble control shRNA (“shSCR”), expression of Nrf2 mRNA (A) and (B) and listed proteins (C) and (D) were shown. Above cells were also irradiated with UV (30 mJ/cm 2 ) and cultured 24 hours, and cell viability was tested by MTT assay (E) and (F) . Nrf2 expression ( vs . loading Erk1/2) was quantified ( C and D ). For each assay, n=5. Experiments in this figure were repeated three times to insure consistency of results. * p <0.05 vs . “shSCR” cells.

Article Snippet: The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours.

Techniques: Expressing, Infection, shRNA, Sequencing, Control, Irradiation, Cell Culture, MTT Assay

Journal: Oncotarget

Article Title: miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

doi: 10.18632/oncotarget.14489

Figure Lengend Snippet: Listed protein and mRNA expressions in stable RPEs (A) and (B) and RGCs (C) and (D) with miR-141 (two lines, “L1/L2”) or scramble miRNA control (“miR-C”) were examined by Western blot assay ( A and C ) and RT-qPCR assay ( B and D ), respectively. Nuclear Nrf2 expression was also shown ( A and C , lower panels). Protein expressions of Nrf2, HO1 and NQO1 ( vs . β-actin) were quantified ( A and C ). For each assay, n=5. Experiments in this figure were repeated four times to insure consistency of results. * p <0.05 vs . “Parental” cells ( A – D ).

Article Snippet: The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours.

Techniques: Control, Western Blot, Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

doi: 10.18632/oncotarget.14489

Figure Lengend Snippet: Expression of miRNA-141 (A) Keap1 mRNA (B) and listed proteins ( B ) in RPEs transfected with antagomiR-141 or non-sense control antagomiR (“antagomiR-C”) were shown. Cells were also subjected to UV radiation (30 mJ/cm 2 ) and cultured for applied time, and cell viability was tested by MTT assay (C) Cell apoptosis was tested by Histone DNA ELISA assay (D) . “Transfection” indicates transfection reagents only. Protein expressions of Keap1, Nrf2, HO1 and NQO1 ( vs . β-actin) were quantified ( A and C ). For each assay, n=5. Experiments in this figure were repeated three times to insure consistency of results. * p <0.05 vs . “antagomiR-C” cells.

Article Snippet: The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours.

Techniques: Expressing, Transfection, Control, Cell Culture, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

doi: 10.18632/oncotarget.14489

Figure Lengend Snippet: miRNA-141-expressing RPEs (“L1/2”) were infected with lentiviral Nrf2 shRNA (“shNrf2-A/-B”, with non-overlapping sequence) or scramble control shRNA (“shSCR”), expression of Nrf2 mRNA (A) and (B) and listed proteins (C) and (D) were shown. Above cells were also irradiated with UV (30 mJ/cm 2 ) and cultured 24 hours, and cell viability was tested by MTT assay (E) and (F) . Nrf2 expression ( vs . loading Erk1/2) was quantified ( C and D ). For each assay, n=5. Experiments in this figure were repeated three times to insure consistency of results. * p <0.05 vs . “shSCR” cells.

Article Snippet: The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours.

Techniques: Expressing, Infection, shRNA, Sequencing, Control, Irradiation, Cell Culture, MTT Assay