Journal: Journal of Thoracic Disease

Article Title: The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells

doi: 10.21037/jtd-20-1362

Figure Lengend Snippet: Effects of UBQLN1 knockdown (KD) on cell viability, proliferation, and apoptosis in A549 cells. (A) Cell viability determined by MTT assay; (B) cell proliferation determined by BrdU assay; (C) cell apoptosis determined by TUNEL assay. Left: representative images showing apoptotic cells; right: quantification of cell apoptosis; (D) representative Western blot images showing apoptosis markers; (E) quantification of cleaved caspase-3 in (D); (F) quantification of protein levels of Bcl-2 and Bax in (D); (G) quantification of Bcl-2/Bax in (D). n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group. UBQLN1, ubiquilin 1; KD, knockdown.

Article Snippet: Cell culture Human non-small cell lung carcinoma cell line A549 (ATCC ® CCL-185 TM ) and H358 (ATCC ® CRL-5807 TM ), and human normal lung epithelial BEAS-2B cells (ATCC ® CRL-9609 TM ) were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Knockdown, MTT Assay, BrdU Staining, TUNEL Assay, Western Blot, Control

Journal: Journal of Thoracic Disease

Article Title: The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells

doi: 10.21037/jtd-20-1362

Figure Lengend Snippet: Effects of p53 overexpression on cell viability, proliferation, and apoptosis in UBQLN1-KD A549 cells. (A) Representative Western blot images showing p53 protein levels; (B) cell viability determined by MTT assay; (C) cell proliferation determined by BrdU assay; (D) cell apoptosis determined by TUNEL assay. Left: representative images showing apoptotic cells; right: quantification of cell apoptosis; (E) representative Western blot images showing apoptosis markers; (F) quantification of cleaved caspase-3 in (E); (G) quantification of Bcl-2/Bax in (E). n=4. ***, P<0.001 vs. siRNA control group; ##, P<0.01 and ###, P<0.001 vs. UBQLN1 KD group. OE, overexpression; UBQLN1, ubiquilin 1; KD, knockdown.

Article Snippet: Cell culture Human non-small cell lung carcinoma cell line A549 (ATCC ® CCL-185 TM ) and H358 (ATCC ® CRL-5807 TM ), and human normal lung epithelial BEAS-2B cells (ATCC ® CRL-9609 TM ) were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Over Expression, Western Blot, MTT Assay, BrdU Staining, TUNEL Assay, Control, Knockdown

Journal: Journal of Thoracic Disease

Article Title: The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells

doi: 10.21037/jtd-20-1362

Figure Lengend Snippet: Effects of UBQLN1 knockdown (KD) on the activities of proteasome and autophagy in A549 cells. (A) Effects of UBQLN1 KD on proteasome activity; (B) representative Western blot images showing the effects of UBQLN1 KD on autophagic markers; (C) quantification of LC3-II/LC3-I in (B); (D) representative Western blot images showing the effects of autophagy inhibitor BFA on LC3-II expression; (E) quantification of autophagic flux based on (D); (F) representative Western blot images showing the effects of autophagy inhibitor BFA on p53 protein levels; (G) quantification of p53 protein levels in (F). n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group; ##, P<0.01 vs. UBQLN1 KD group. UBQLN1, ubiquilin 1; BFA, brefeldin A.

Article Snippet: Cell culture Human non-small cell lung carcinoma cell line A549 (ATCC ® CCL-185 TM ) and H358 (ATCC ® CRL-5807 TM ), and human normal lung epithelial BEAS-2B cells (ATCC ® CRL-9609 TM ) were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Knockdown, Activity Assay, Western Blot, Expressing, Control

Journal: Journal of Thoracic Disease

Article Title: The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells

doi: 10.21037/jtd-20-1362

Figure Lengend Snippet: Effects of UBQLN1 knockdown (KD) on ER stress, ROS formation, and the mTOR signaling in A549 cells. (A) Representative Western blot images showing proteins markers of ER stress; (B) representative images showing real time formation of intracellular ROS (magnification ×400); (C) quantification of ROS formation in (B). (D) representative Western blot images showing phosphorylation of mTOR and its downstream S6K; (E) quantification of phosphorylation levels of mTOR and S6K in (C); (F) schematic diagram of UBQLN1 on cell viability, proliferation, and apoptosis in A549 cancer cells. UBQLN1 impairs mTORC1 activity leading to the activation of autophagy and subsequent degradation of p53. Loss of p53 will affect cell viability, proliferation, and apoptosis. n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group; #, P>0.05 vs. siRNA control group. UBQLN1, ubiquilin 1.

Article Snippet: Cell culture Human non-small cell lung carcinoma cell line A549 (ATCC ® CCL-185 TM ) and H358 (ATCC ® CRL-5807 TM ), and human normal lung epithelial BEAS-2B cells (ATCC ® CRL-9609 TM ) were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Knockdown, Western Blot, Phospho-proteomics, Activity Assay, Activation Assay, Control

Journal: Nature Communications

Article Title: Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

doi: 10.1038/s41467-022-31386-1

Figure Lengend Snippet: Schematic representation of BCR-ABL1 fusion chromosome development ( a ). K562-fLUC cell death following pDNA electroporation. Cell viability was determined via bioluminescence signal intensity, wherein the value of total flux (p/s) bioluminescence of pcDNA3 electroporated cells was set to 100%. Cell death was determined 72 h post electroporation ( b ). Indel mutation quantification measured via the T7E1 assay in K562 cells edited with CCExo targeting the BCR-ABL1 region 72 h post electroporation ( c ). Data represent three independent experiments ( n = 3). * P < 0.05, **< 0.01, ***< 0.001 and **** P < 0.0001. All P values are from ordinary one-way ANOVA followed by Tukey’s multiple comparisons test compared to Cas9 values. Data are presented as mean values ± SEM as appropriate ( b – c ). PBMCs of CML patients showed enhanced cell death following CCExo RNP electroporation. Cell death was determined by TUNEL assay, where cell death was normalized to an apoptosis induced control cell sample. Dots represent values corresponding to individual patient donor PBMCs ( n = cells from two CML patients) ( d ). Indel mutation quantification measured via the T7E1 assay in PBMCs of CML patients edited with CCExo targeting the BCR-ABL1 region 72 h after the electroporation. Data represent two independent patient donor PBMCs ( n = 2). ** P < 0.01. All P values are from ordinary one-way ANOVA followed by Tukey’s multiple comparisons test compared to Cas9 values. Data are presented as mean values ± SEM as appropriate ( e ). Diagram showing the timeline of in vivo experiments using SCID mice with xenograft K562 cancer model for potential anti-tumor therapeutic application determination. At day 18, pDNA (50 μg/animal) was intratumorally electroporated. At day 50, the experiment was terminated ( f ). Cumulative representation of tumor volumes, treated with indicated plasmids. Data are presented as mean values ± SEM as appropriate. All P values are from unpaired t test with Welch’s correction, ( n = 5 animals). * P < 0.05, **< 0.01 and **** P < 0.0001 compared to CCExo treated animals ( g ). Survival plot of animals involved in the experiment ( h ). Tumor size of K562 xenograft tumors, taken from the mice at the end of the experiments ( i ). TUNEL assay determining apoptotic cell death (green-Brdu-Red positive cells, blue-DAPI stain) in tumors tissue slides, derived from animals, treated with CCExo. A representative image from individual treated animal is provided. Scale bar, 50 μm ( j ).

Article Snippet: When tumors reached defined size (40 mm 3 ), 50 μg of plasmid DNA, dissolved in 150 mM NaCl was injected and afterwards electroporated using Gene Pulser Xcell TM Electroporation System (Biorad; settings: 900 V, 100μs, 6 pulses in 1 s interval).

Techniques: Electroporation, Mutagenesis, TUNEL Assay, Control, In Vivo, Staining, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Pinocembrin ameliorates lipopolysaccharide-induced HK-2 cell apoptosis and inflammation by regulating endoplasmic reticulum stress

doi: 10.3892/etm.2022.11440

Figure Lengend Snippet: PINO exerts effects on inflammation, oxidative stress and apoptosis in LPS-induced HK-2 cells by regulating ERS. HK-2 cells were treated with TM (1 µg/ml) for 2 h prior to PINO treatment and LPS stimulation. (A) Protein expression levels of TNF-α, IL-6 and IL-1β were measured using western blotting. The concentrations of (B) MDA and (C) GSH in the supernatant of the culture media were determined using their corresponding commercial kits. (D) TUNEL assay was performed to assess apoptosis. (E) Quantification of the apoptosis rate. (F) Apoptosis-associated proteins were detected using western blotting. *** P<0.005 vs. control; ## P<0.01, ### P<0.001 vs. the LPS group; Δ P<0.05, ΔΔΔ P<0.001 vs. the LPS + PINO group. PINO, pinocembrin; LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor-α; GSH, glutathione; MDA, malondialdehyde; ERS, endoplasmic reticulum stress; TM, tunicamycin.

Article Snippet: In addition, tunicamycin (TM; MedChemExpress), an agonist of endoplasmic reticulum stress (ERS), was introduced into HK-2 cells at a concentration of 1 µg/ml at 37˚C at 2 h prior to 200 µg/ml PINO treatment to explore the regulatory mechanism of PINO.

Techniques: Expressing, Western Blot, TUNEL Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Honokiol inhibits endoplasmic reticulum stress-associated lipopolysaccharide-induced inflammation and apoptosis in bovine endometrial epithelial cells

doi: 10.3892/etm.2021.10911

Figure Lengend Snippet: Honokiol treatment improves the viability of LPS-treated bEECs. (A) Chemical structural formula of HKL. (B) The viability of bEECs following treatment with different doses of HKL as detected by MTT. (C) The viability of LPS-stimulated bEECs following treatment with different doses of HKL as detected by MTT. ** P<0.01 and *** P<0.001 vs. Control; ## P<0.01 vs. LPS. HKL or Hon, honokiol; LPS, lipopolysaccharide; bEECs, bovine endometrial epithelial cells.

Article Snippet: BEND bovine endometrial epithelial cells (bEECs; ATCC ® CRL-2398 TM ) were acquired from American Type Culture Collection.

Techniques: Control

Journal: Experimental and Therapeutic Medicine

Article Title: Honokiol inhibits endoplasmic reticulum stress-associated lipopolysaccharide-induced inflammation and apoptosis in bovine endometrial epithelial cells

doi: 10.3892/etm.2021.10911

Figure Lengend Snippet: Honokiol treatment inhibits LPS-induced inflammation and apoptosis in bEECs. (A) The expression levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 in LPS-stimulated bEECs following treatment with different doses of HKL as detected by ELISA. (B) The mRNA expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 in LPS-stimulated bEECs following treatment with different doses of HKL as detected by reverse transcription-quantitative PCR. (C) Apoptosis level of LPS-stimulated bEECs following treatment with different doses of HKL as observed by TUNEL staining, (D) which was quantified. (E) The expression of apoptosis markers Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9 in LPS-stimulated bEECs following treatment with different doses of HKL as detected by western blotting, (F) which was quantified. * P<0.05, ** P<0.01, *** P<0.001 vs. Control. # P<0.05, ## P<0.01, ### P<0.001 vs. LPS. HKL or Hon, honokiol; LPS, lipopolysaccharide; bEECs, bovine endometrial epithelial cells.

Article Snippet: BEND bovine endometrial epithelial cells (bEECs; ATCC ® CRL-2398 TM ) were acquired from American Type Culture Collection.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Honokiol inhibits endoplasmic reticulum stress-associated lipopolysaccharide-induced inflammation and apoptosis in bovine endometrial epithelial cells

doi: 10.3892/etm.2021.10911

Figure Lengend Snippet: Honokiol inhibits LPS-induced ER stress in bEECs. The expression of ER stress-related proteins in LPS-stimulated bEECs following treatment with different doses of HKL as detected by western blotting. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. ## P<0.01, ### P<0.001 vs. LPS. HKL or Hon, honokiol; LPS, lipopolysaccharide; bEECs, bovine endometrial epithelial cells; ATF6, activating transcription factor 6; CHOP, CCAAT-enhancer-binding protein homologous protein; IRE1, inositol-requiring enzyme 1; c-, cleaved.

Article Snippet: BEND bovine endometrial epithelial cells (bEECs; ATCC ® CRL-2398 TM ) were acquired from American Type Culture Collection.

Techniques: Expressing, Western Blot, Control, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Honokiol inhibits endoplasmic reticulum stress-associated lipopolysaccharide-induced inflammation and apoptosis in bovine endometrial epithelial cells

doi: 10.3892/etm.2021.10911

Figure Lengend Snippet: Honokiol mitigates ER stress to inhibit LPS-induced inflammation and apoptosis in bEECs. (A) The expression level of proinflammatory cytokines TNF-α, IL-1β and IL-6 in LPS-stimulated bEECs treated with 20 µM HKL in the absence or presence of tunicamycin as detected by ELISA. (B) The mRNA expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 in LPS-stimulated bEECs treated with HKL in the absence or presence of tunicamycin as detected by reverse transcription-quantitative PCR. (C) The apoptosis level of LPS-stimulated bEECs treated with HKL in the absence or presence of tunicamycin as observed by TUNEL staining, (D) which was quantified. (E) The expression level of apoptosis-related proteins Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9 in LPS-stimulated bEECs treated with HKL in the absence or presence of tunicamycin as detected by western blotting, (F) which was quantified. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. # P<0.05, ## P<0.01, ### P<0.001 vs. the LPS group. & P<0.01, && P<0.001 vs. the Hon + LPS group. HKL or Hon, honokiol; LPS, lipopolysaccharide; bEECs, bovine endometrial epithelial cells; tun, tunicamycin.

Article Snippet: BEND bovine endometrial epithelial cells (bEECs; ATCC ® CRL-2398 TM ) were acquired from American Type Culture Collection.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot, Control

Journal: bioRxiv

Article Title: Parthanatos-inducing zinc agent C010DS-Zn elicits anti-tumor immune responses involving T cells and macrophages in vivo

doi: 10.1101/2021.03.18.433812

Figure Lengend Snippet: Basic substance information on C010DS-Zn, and its control substances C005D-Zn, zinc sulfate (ZnSO 4 ), and “Free Zn-Pyr (7:1)” (the mixture of zinc sulfate and sodium pyrithione at zinc-to-pyrithione molar ratio of 7-to-1). (A) Molecular structure of C010DS, 34 kDa (M w ) γPGA polymer each conjugated with 1 Cy5.5 label, 2 PEG-folate sidechains, 2 PEG-cRGDfK sidechains, and 10 PEG-S-S-pyrithione sidechains. C010DS-Zn preparation is described in the Methods. (B) Molecular structure of C005D, 34 kDa (M w ) γPGA polymer each conjugated with 1 Cy5.5 label, 3 PEG-folate sidechains, and 3 PEG-cRGDfK sidechains. C005D-Zn preparation is described in the Methods. (C) Comparative in vitro cytotoxicity evaluation on the tested compounds using LDH release assays after 24h treatments against 4T1 cells. (D) Results of the in vitro time-resolved apoptosis-necrosis flow cytometry assay at fixed doses against 4T1 cells using A5 and PI labels. (E) Results of the in vitro dose-resolved apoptosis-necrosis flow cytometry assay at 3h time point against 4T1 cells using A5 and PI labels. (F) In vitro PAR-ELISA assay results on the C010DS-Zn treated 4T1 cells with or without the PARP inhibitor PJ34. * p <0.05. ¶ Indicated group displayed significantly higher PAR signal than the Control, 2.5 μM Zn, or 5 μM Zn C010DS-Zn treatment groups at p <0.05 with or without PJ34 co-treatment.

Article Snippet: Mouse mammary carcinoma 4T1 cells (ATCC ® CRL-2539 TM ) and mouse colorectal carcinoma CT26 cells (ATCC ® CRL-2638 TM ) were cultured in RPMI Medium 1640 (61870-036, Gibco) supplemented with 10% Foetal Bovine Serum (FBS; HyClone, SH3007103) and 1% Penicillin-Streptomycin (P/S; Sigma Aldrich, P4333).

Techniques: Control, Polymer, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Parthanatos-inducing zinc agent C010DS-Zn elicits anti-tumor immune responses involving T cells and macrophages in vivo

doi: 10.1101/2021.03.18.433812

Figure Lengend Snippet: In vitro attenuation of 1.5h C010DS-Zn treatment effects against 4T1 including nuclear AIF translocation, DNA fragmentation, and cell viability loss by PARP inhibitor PJ34. (A) Representative confocal fluorescence images of the 4T1 cells treated for 1.5h with vehicle, ZnSO4, C005D-Zn, or C010DS-Zn at different concentrations, labelled with Hoechst (blue: adherent cell count), TUNEL (green: DNA breaks), and anti-AIF (red), with or without the PARP inhibitor PJ34 co-incubation. Bar=100μm. (B) Quantitative analysis of the fluorescence images for cell viability, nuclear AIF translocation, and nuclear TUNEL intensity using CirAvgInten function. 3 imaging fields quantified for each group (n=3). * p <0.05. ** p <0.005. *** p <0.0005. ¶Indicated group’s average value is significantly greater than those of all other treatment groups minimally at p <0.05. C010DS-Zn at 60 μM Zn treatment group resulted in complete cell loss, and hence its quantitative imaging analyses could not be performed.

Article Snippet: Mouse mammary carcinoma 4T1 cells (ATCC ® CRL-2539 TM ) and mouse colorectal carcinoma CT26 cells (ATCC ® CRL-2638 TM ) were cultured in RPMI Medium 1640 (61870-036, Gibco) supplemented with 10% Foetal Bovine Serum (FBS; HyClone, SH3007103) and 1% Penicillin-Streptomycin (P/S; Sigma Aldrich, P4333).

Techniques: In Vitro, Translocation Assay, Fluorescence, Cell Counting, TUNEL Assay, Incubation, Imaging

Journal: bioRxiv

Article Title: Parthanatos-inducing zinc agent C010DS-Zn elicits anti-tumor immune responses involving T cells and macrophages in vivo

doi: 10.1101/2021.03.18.433812

Figure Lengend Snippet: In vivo characterization of the anti-tumor responses induced by C010DS-Zn intravenous treatments against 4T1 murine TNBC model on Balb/c mice and against CT26 murine CRC model on Balb/c mice. (A) 4T1-Balb/c treatment model scheme, tumor growth kinetics, and notable immune responses in the TME. (B) CT26-Balb/c treatment model scheme, tumor growth kinetics, and notable immune responses in the TME. * p <0.05. ** p <0.005. *** p <0.0005.

Article Snippet: Mouse mammary carcinoma 4T1 cells (ATCC ® CRL-2539 TM ) and mouse colorectal carcinoma CT26 cells (ATCC ® CRL-2638 TM ) were cultured in RPMI Medium 1640 (61870-036, Gibco) supplemented with 10% Foetal Bovine Serum (FBS; HyClone, SH3007103) and 1% Penicillin-Streptomycin (P/S; Sigma Aldrich, P4333).

Techniques: In Vivo

Journal: bioRxiv

Article Title: Parthanatos-inducing zinc agent C010DS-Zn elicits anti-tumor immune responses involving T cells and macrophages in vivo

doi: 10.1101/2021.03.18.433812

Figure Lengend Snippet: Rat plasma pharmacokinetic profiling of an intravenously dosed C010DS-Zn bolus and the in vivo effect of “sub-anticancer” C010DS-Zn dosing against the macrophages in the TME. (A) Plasma pharmacokinetic profile of C010DS-Zn that separately traced C010DS via Cy5.5 signal and zinc levels after a single intravenous bolus injection of C010DS-Zn using Sprague-Dawley Rats. Time-resolved excess plasma zinc post the C010DS-Zn injection is also plotted in % units of the total zinc injected using C010DS-Zn. (B) 13 days non-anticancer dosing scheme against 4T1-Balb/c model, its tumor growth kinetics, and the macrophage immune response to the treatment in the collected tumors. (C) 16 days non-anticancer dosing scheme against 4T1-Balb/c model, its tumor growth kinetics, and the macrophage immune response to the treatment in the collected tumors. * p <0.05.

Article Snippet: Mouse mammary carcinoma 4T1 cells (ATCC ® CRL-2539 TM ) and mouse colorectal carcinoma CT26 cells (ATCC ® CRL-2638 TM ) were cultured in RPMI Medium 1640 (61870-036, Gibco) supplemented with 10% Foetal Bovine Serum (FBS; HyClone, SH3007103) and 1% Penicillin-Streptomycin (P/S; Sigma Aldrich, P4333).

Techniques: Clinical Proteomics, In Vivo, Injection

Journal: Technology in Cancer Research & Treatment

Article Title: TNFRSF10B, a Therapeutic Target for Oral Squamous Cell Carcinoma Through Integrated Bioinformatics and Preliminary Experiments

doi: 10.1177/15330338261426318

Figure Lengend Snippet: TNFRSF10B expression validation and functional characterization. (A–B) Detection of TNFRSF10B protein abundance by Western blot and its quantitative assessment. (C) RT-qPCR assessment of TNFRSF10B mRNA levels. (D–E) Protein changes in SCC-4 cells after TNFRSF10B silencing, examined by Western blot and quantified accordingly. (F) RT-qPCR detection of TNFRSF10B mRNA expression in SCC-4 cells. (G) Results of CCK-8 assay for cell viability following TNFRSF10B knockdown in SCC-4 cells, **P < .01, ***P < .001, compared with control groups (n = 3). (H-I) Protein expression patterns in SCC-9 cells following TNFRSF10B knockdown, analyzed and quantified by Western blot. (J) RT-qPCR measurement of TNFRSF10B transcript level in SCC-9 cells. (K) Results of CCK-8 assay for cell viability following TNFRSF10B knockdown in SCC-9 cells, **P < .01, ***P < .001, compared with control groups (n = 3).

Article Snippet: SCC-4 (ATCC ® CRL-1624 TM , RRID: CVCL_1684) and SCC-9 (ATCC ® CRL-1629 TM , RRID: CVCL_1685) human OSCC cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Biomarker Discovery, Functional Assay, Quantitative Proteomics, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Knockdown, Control

Journal: Technology in Cancer Research & Treatment

Article Title: TNFRSF10B, a Therapeutic Target for Oral Squamous Cell Carcinoma Through Integrated Bioinformatics and Preliminary Experiments

doi: 10.1177/15330338261426318

Figure Lengend Snippet: Pro-apoptotic effects of TNFRSF10B knockdown in OSCC cells confirmed by flow cytometry and TUNEL staining. (A-B) Flow cytometry analysis and quantification of apoptosis in SCC-4 cells (A) and SCC-9 cells (B) following TNFRSF10B knockdown. (C-D) TUNEL staining-based quantification of apoptosis in SCC-4 cells. Scale bar: 200 μm; ***P < .001, compared with si-NC control (n = 3).

Article Snippet: SCC-4 (ATCC ® CRL-1624 TM , RRID: CVCL_1684) and SCC-9 (ATCC ® CRL-1629 TM , RRID: CVCL_1685) human OSCC cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Flow Cytometry, TUNEL Assay, Staining, Control

Journal: Technology in Cancer Research & Treatment

Article Title: TNFRSF10B, a Therapeutic Target for Oral Squamous Cell Carcinoma Through Integrated Bioinformatics and Preliminary Experiments

doi: 10.1177/15330338261426318

Figure Lengend Snippet: TNFRSF10B knockdown reshapes apoptotic signaling by modulating core pathway proteins. (A-B) Caspase-3, PARP, Bcl-2, and Bax expression levels were assessed by western blotting in SCC-4 (A) and SCC-9 (B) cells. **P < .01, ***P < .001.

Article Snippet: SCC-4 (ATCC ® CRL-1624 TM , RRID: CVCL_1684) and SCC-9 (ATCC ® CRL-1629 TM , RRID: CVCL_1685) human OSCC cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Expressing, Western Blot

Journal: Theranostics

Article Title: Tumor-suppressing miR-141 gene complex-loaded tissue-adhesive glue for the locoregional treatment of hepatocellular carcinoma

doi: 10.7150/thno.24056

Figure Lengend Snippet: Cytotoxicity of NPX and NPX-glue with various N/P ratios. (A) Hep3B cells were plated at a density of 5×10 4 cells/well in 24-well plates on day 0. On day 1, the cells were treated with NCmiR:NPX with the indicated N/P ratios. The cell viability was determined on day 3. The cells transfected with Lipofectamine 2000 were the control (CTR) for this experiment. (B) Cells were treated with NCmiR:NPX (N/P ratio = 1.6) or NCmiR:NPX-glue (N/P ratio = 1.6) and their % cell viabilities were compared with that of the untreated cells, which were regarded as 100%. The values are mean ± S.D. calculated from three wells. * indicates a statistical significance of p < 0.05 using Student's t -test. The experiments were repeated three times.

Article Snippet: Hep3B human hepatocellular carcinoma cells (ATCC HB-8064 TM ) were purchased directly from ATCC at the beginning of the experiments and cultured in DMEM supplemented with 10% FBS, and 1× penicillin/streptomycin, and incubated at 37 oC under 5% CO 2 .

Techniques: Transfection, Control

Journal: Theranostics

Article Title: Tumor-suppressing miR-141 gene complex-loaded tissue-adhesive glue for the locoregional treatment of hepatocellular carcinoma

doi: 10.7150/thno.24056

Figure Lengend Snippet: Gene delivery efficiency of NPX-glue into Hep3B cells . (A) Hep3B cells were plated on a coverslip in a 24-well plate on day 0. The blank NPX-glue or Cy5 NCmiR:NPX-glue were placed on a side of the well under the culture media. The coverslips with the seeded Hep3B cells were placed in the well on day 1. Fluorescence from Cy5 was observed on days 6 and 14. Transfection of Cy5 NCmiR using G-fectin was used as a control ( Cy5 NCmiR-Gfectin) and the image was taken 24 h after transfection (Day 1*). (B) The blank NPX-glue or pEGFP:NPX-glue was placed on a side of the well under the culture media. The coverslips with the seeded Hep3B cells were placed in the well on day 1. The fluorescence from GFP was observed on days 6 and 14. Transfection of pEGFP using Lipofectamine 2000 was used as a control (pEGFP:LF) and the image was taken 24 h after transfection (Day 1*). 10X magnification (Scale bar) (C) Percentage of cells that expressed Cy5 NCmiR on days 6 and 14. (D) Percentage of cells that expressed GFP on days 6 and 14. The values are the mean ± S.D. of three wells. The experiments were repeated three times.

Article Snippet: Hep3B human hepatocellular carcinoma cells (ATCC HB-8064 TM ) were purchased directly from ATCC at the beginning of the experiments and cultured in DMEM supplemented with 10% FBS, and 1× penicillin/streptomycin, and incubated at 37 oC under 5% CO 2 .

Techniques: Fluorescence, Transfection, Control

Journal: Theranostics

Article Title: Tumor-suppressing miR-141 gene complex-loaded tissue-adhesive glue for the locoregional treatment of hepatocellular carcinoma

doi: 10.7150/thno.24056

Figure Lengend Snippet: Expression levels of the target genes in the cells treated with miR-141:NPX-glue. (A) Cellular expression level of miR-141 when Blank NPX-glue, NCmiR:NPX-glue, or miR-141:NPX-glue was placed on the side of the well in a 6-well plate. Coverslips seeded with Hep3B cells were placed into the wells on day 1. On day 3, the cells were harvested, total RNA was extracted from the cells, and qPCR was performed to measure the expression level of miR-141. (B) Schematic drawing of inhibition of the target genes by miR-141. (C) The mRNA expression levels of MAP4K4 , TM4SF1 , KEAP1 , HDGF , and TIAM1 . The mRNA expression levels were normalized against GAPDH and the relative expression levels of each mRNA were compared with those of blank NPX-glue-treated cells. The values are the mean ± S.D. of three wells. * indicates a statistical significance of p < 0.05 using Student's t -test. The experiments were repeated three times.

Article Snippet: Hep3B human hepatocellular carcinoma cells (ATCC HB-8064 TM ) were purchased directly from ATCC at the beginning of the experiments and cultured in DMEM supplemented with 10% FBS, and 1× penicillin/streptomycin, and incubated at 37 oC under 5% CO 2 .

Techniques: Expressing, Inhibition

Journal: Theranostics

Article Title: Tumor-suppressing miR-141 gene complex-loaded tissue-adhesive glue for the locoregional treatment of hepatocellular carcinoma

doi: 10.7150/thno.24056

Figure Lengend Snippet: Tumor-suppressive effect of miR-141:NPX-glue. (A) Hep3B cells (5×10 4 cells) were injected subcutaneously (SQ) into the flank of mice. When the tumor size reached 250 mm 3 , NCmiR:NPX-glue or miR-141:NPX-glue was injected into the tumors thrice. The volumes of the tumors were monitored for 24 days. (B) The tumors were harvested 24 days after injection and their weight was measured. The values are the mean ± S.D. of three tumors. (C) Body weights of the mice were monitored during the experiment. The values are the mean ± S.D. of five mice. (D) H&E and TUNEL staining of the tumor tissues treated with NCmiR:NPX-glue or miR-41:NPX-glue. The yellow arrow indicates apoptotic area. (E) Immunoblot analysis of MAP4K4, TIAM1, BCL-2, cleaved caspase 3, and β-actin in the tissues treated with NCmiR:NPX-glue (lanes 1 and 2) and miR-141:NPX-glue (lanes 3 and 4). Values of the band intensity (in red characters) measured by Image J are shown below the figures. ** indicates a statistical significance of p < 0.05 using Student's t -test. The experiments were repeated three times.

Article Snippet: Hep3B human hepatocellular carcinoma cells (ATCC HB-8064 TM ) were purchased directly from ATCC at the beginning of the experiments and cultured in DMEM supplemented with 10% FBS, and 1× penicillin/streptomycin, and incubated at 37 oC under 5% CO 2 .

Techniques: Injection, TUNEL Assay, Staining, Western Blot

Journal: Scientific Reports

Article Title: Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4

doi: 10.1038/s41598-017-09936-1

Figure Lengend Snippet: Protein complexes differentially expressed in the subcellular fractions of control and B[a]P-exposed RT4 cells. ( A ) 2D BN/SDS-PAGE gel from the cytosolic, membrane/organelle ( B ), and nuclear fraction ( C ) of RT4 cells exposed to B[a]P (0.5 µM, 24 h). Samples from control and B[a]P-exposed cells were separated and compared by using the Delta2D v4 software. Identified proteins with statistically significant alteration differences between control and exposed cells were marked with arrows. Protein spots of interest were excised, trypsin-digested, and subjected to analysis by MALDI-TOF-MS. The identified proteins are listed with their ID numbers in Suppl. Tables , and .

Article Snippet: The human bladder cancer cell line RT4 (ATCC ® HTB-2 TM ) was cultured until confluency in McCoy’s 5 A medium, supplemented with 10% fetal bovine serum, 7.4 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Control, SDS Page, Membrane, Software

Journal: Scientific Reports

Article Title: Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4

doi: 10.1038/s41598-017-09936-1

Figure Lengend Snippet: Evaluation of the cytotoxic potential of B[a]P in RT4 cells: To determine the potential of B[a]P to initiate cytotoxic effects in human bladder epithelial cells, various endpoints were measured, including formation of reactive oxygen species (ROS, A ), apoptosis (TUNEL assay, B ), cell proliferation (EdU, C ). Cells were cultured on 96-well plates with a clear bottom and were exposed to different concentrations of B[a]P (0.1 to 100 µM) for 24 h. H 2 O 2 (100 µM) was used as positive control in ROS assay, while a TUNEL-positive control was obtained by incubation with DNase I. In control, cells were exposed to DMSO (<0.1%). The data is presented as mean ± standard deviation of four independent experiments with RT4 cell lysates. The level of significance relative to the control was determined by using the t-test (*p < 0.05, ***p < 0.001).

Article Snippet: The human bladder cancer cell line RT4 (ATCC ® HTB-2 TM ) was cultured until confluency in McCoy’s 5 A medium, supplemented with 10% fetal bovine serum, 7.4 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: TUNEL Assay, Cell Culture, Positive Control, ROS Assay, Incubation, Control, Standard Deviation

Journal: Scientific Reports

Article Title: Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4

doi: 10.1038/s41598-017-09936-1

Figure Lengend Snippet: Estimation of cellular cofactors (NADPH and NADPt) and the involved enzymes glucose-6-phosphate dehydrogenase (G6PD) and glucose-6-phosphat-isomerase (GPI). ( A ) Schematic representation of enzymes involved in redirection of the flux from glycolysis to Pentose Phosphate Pathway (PPP). The rerouting of glucose into oxidative PPP depends on a rapid increase of the G6PD flux, which can generate strong inhibitor of GPI thus facilitating a forward flux into PPP. ( B ) Analysis of G6PD and GPI enzyme activity: The enzyme assay was carried out in a 96-well plate in cells exposed to B[a]P (0.5 µM, 24 h). NADPH (100 µM) was used as an inhibitor for G6PD, while 6-PG (5 mM) was used as an inhibitor for GPI enzyme assay. Effects of 0.5 µM B[a]P are shown on cellular ( C , D ) and cytosolic ( E , F ) NADPH/NADP + ratio and NADPH content of RT4 cells. The data is presented as mean ± standard error of the mean of four independent experiments. The level of significance relative to the control was determined by using the Anova test (*p < 0.05, ***p < 0.001).

Article Snippet: The human bladder cancer cell line RT4 (ATCC ® HTB-2 TM ) was cultured until confluency in McCoy’s 5 A medium, supplemented with 10% fetal bovine serum, 7.4 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Activity Assay, Enzymatic Assay, Control

Journal: Scientific Reports

Article Title: Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4

doi: 10.1038/s41598-017-09936-1

Figure Lengend Snippet: Significantly up- or down-regulated metabolites extracted from the RT4 cells with methanol (n = 8) after 24 h exposure of the cells to 0.5 µM B[a]P.

Article Snippet: The human bladder cancer cell line RT4 (ATCC ® HTB-2 TM ) was cultured until confluency in McCoy’s 5 A medium, supplemented with 10% fetal bovine serum, 7.4 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques:

Journal: Scientific Reports

Article Title: Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4

doi: 10.1038/s41598-017-09936-1

Figure Lengend Snippet: Significantly up- or down-regulated metabolites extracted from the RT4 cells with n -hexane/methyl tert -butyl ether (n = 8) after 24 h exposure of the cells to 0.5 µM B[a]P.

Article Snippet: The human bladder cancer cell line RT4 (ATCC ® HTB-2 TM ) was cultured until confluency in McCoy’s 5 A medium, supplemented with 10% fetal bovine serum, 7.4 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques:

Journal: Scientific Reports

Article Title: Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4

doi: 10.1038/s41598-017-09936-1

Figure Lengend Snippet: List of altered glycolytic and pentose phosphate pathway-associated proteins (≥2) after B[a]P exposure (0.5 µM, 24 h) compared to controls.

Article Snippet: The human bladder cancer cell line RT4 (ATCC ® HTB-2 TM ) was cultured until confluency in McCoy’s 5 A medium, supplemented with 10% fetal bovine serum, 7.4 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Control

Journal: Cell Death & Disease

Article Title: RPL22L1, a novel candidate oncogene promotes temozolomide resistance by activating STAT3 in glioblastoma

doi: 10.1038/s41419-023-06156-6

Figure Lengend Snippet: A The expressions of endogenous RPL22L1 protein in T98G, A172, LN229 and U251 were detected by Western Blot, GAPDH was used as the internal reference. B T98G and LN229 cell lines were infected with RPL22L1 lentivirus, vector was the control group. After 72 h, Western Blot was used to detect the expressions of RPL22L1 protein. C , D RPL22L1 expressions were efficiently knocked down by two targeted shRNAs (sh1 and sh3) in U251 and A172 cell lines detected by Western Blot, NC served as negative control. E , F The effects of RPL22L1 on the viability of GBM cells were detected by MTS assay ( n = 6). G , H The effects of RPL22L1 on the viability of GBM cells were detected by CCK8 assay ( n = 6). I , J Wound healing assay was used to detect the cell migration abilities of cells (magnification×40, scale bar = 50 μm). K , L Transwell migration and invasion assays were performed to examine the effects of RPL22L1 on migration ( K ) and invasion ( L ) of GBM cells (left, magnification×100, scale bar = 100 μm). All data were shown as mean ± SD of three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t test).

Article Snippet: Human GBM cell lines A-172 (CRL-1620 TM ), LN-229 (CRL-2611 TM ) and T98G (CRL-1690 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Infection, Plasmid Preparation, Control, Negative Control, MTS Assay, CCK-8 Assay, Wound Healing Assay, Migration

Journal: Cell Death & Disease

Article Title: RPL22L1, a novel candidate oncogene promotes temozolomide resistance by activating STAT3 in glioblastoma

doi: 10.1038/s41419-023-06156-6

Figure Lengend Snippet: A Western Blot analysis of the effects of RPL22L1 on the protein levels of p-EGFR, EGFR, p-STAT3 and STAT3. B IHC staining of RPL22L1, p-EGFR and p-STAT3 in normal brain tissues and GBM brain tissues in TMAs (up, magnification×400, scale bar = 50 μm). According to the staining scores to analyze the correlations between RPL22L1 and p-EGFR, p-STAT3 protein expressions (down, Spearman correlation analysis, n = 53). C – G Cells were treated with Gefitinib (10 μmol/L) or Stattic (5 μmol/L) for 48 h. C Western Blot detected the expressions of RPL22L1, EGFR, p-EGFR, STAT3 and p-STAT3, GAPDH was as the internal control. D , E MTS assay and CCK8 assay detected the effects of Gefitinib or Stattic on the viability of T98G-RPL22L1/T98G-Vec cells ( D ) and LN229-RPL22L1/LN229-Vec cells ( E ). All data were shown as mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01; *** P < 0.001, Student’s t test). F , G Transwell migration assays detected the effects of Gefitinib or Stattic on the migration of T98G-RPL22L1/T98G-Vec cells ( F ) and LN229-RPL22L1/LN229-Vec cells ( G ). H , I Transwell invasion assays detected the effects of Gefitinib or Stattic on the invasion of T98G-RPL22L1/T98G-Vec cells ( H ) and LN229-RPL22L1/LN229-Vec cells ( I ). Representative images were shown (left, magnification×100, scale bar = 100 μm). All data were shown as mean ± SD of three independent experiments (right, * P < 0.05, ** P < 0.01; *** P < 0.001, Student’s t test). J Western Blot was used to detect the expressions of E-cadherin, β-catenin, N-cadherin, Vimentin, α-SMA, Snail2 and Twist1 in T98G-RPL22L1/T98G-Vec cells, GAPDH was the internal control. K , L IF assay detected the expressions of N-cadherin ( K ) and Vimentin ( L ) protein (left, red: N-cadherin and Vimentin, blue: cell nucleus, magnification×200, scale bar = 50 μm). Statistics of relative IF value of Gefitinib and Stattic on the expressions of N-cadherin and Vimentin in T98G-RPL22L1/T98G-Vec cells. All data were shown as mean ± SD of three independent experiments (right, * P < 0.05, Student’s t test).

Article Snippet: Human GBM cell lines A-172 (CRL-1620 TM ), LN-229 (CRL-2611 TM ) and T98G (CRL-1690 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Immunohistochemistry, Staining, Control, MTS Assay, CCK-8 Assay, Migration

Journal: Cell Death & Disease

Article Title: RPL22L1, a novel candidate oncogene promotes temozolomide resistance by activating STAT3 in glioblastoma

doi: 10.1038/s41419-023-06156-6

Figure Lengend Snippet: A–F GBM cells were treated with TMZ (7.5 μmol/L), Gefitinib (10 μmol/L), Stattic (5 μmol/L), TMZ+Gefitinib, TMZ+Stattic for 48 h, DMSO was the negative control. A, B MTS experiment and CCK8 assay were performed to detect proliferation of T98G-RPL22L1/T98G-Vec cells ( A ) and LN229-RPL22L1/LN229-Vec cells ( B ). C , D T98G-RPL22L1/T98G-Vec cells ( C ) and LN229-RPL22L1/LN229-Vec cells ( D ) were treated at the specified concentration for 48 h and IC50 were determined by MTS and CCK8 methods. E , F Colony formation tests of T98G-RPL22L1/T98G-Vec cells ( E ) and LN229-RPL22L1/LN229-Vec cells ( F ). All data were shown as mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test). G–I LN229-RPL22L1/LN229-Vec cells were injected subcutaneously into the right shoulder of nude mice. One week later, intraperitoneal injection of TMZ (60 mg/kg), TMZ+Stattic (5 mg/kg) to nude mice bearing tumors for 13 days. After drug treatment, the subcutaneous tumors were taken, photographed ( G ) and weighed ( I ). The tumor size ( H ) was monitored every other day and statistics were performed ( n = 4, * P < 0.05, Student’s t test).

Article Snippet: Human GBM cell lines A-172 (CRL-1620 TM ), LN-229 (CRL-2611 TM ) and T98G (CRL-1690 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Negative Control, CCK-8 Assay, Concentration Assay, Injection

Journal: Cell Death & Disease

Article Title: RPL22L1, a novel candidate oncogene promotes temozolomide resistance by activating STAT3 in glioblastoma

doi: 10.1038/s41419-023-06156-6

Figure Lengend Snippet: A–C T98G-RPL22L1/T98G-Vec cells were treated with TMZ (7.5 μmol/L), Gefitinib (10 μmol/L), Stattic (5 μmol/L), TMZ+Gefitinib, TMZ+Stattic for 48 h, DMSO was the negative control. A The cell apoptosis assay was performed with flow cytometry in T98G-RPL22L1/T98G-Vec cells. The histogram displayed the statistics of apoptosis assay, respectively. B The TUNEL assay of T98G-RPL22L1/T98G-Vec cells (left, green: TUNEL, blue: cell nucleus, magnification × 200, scale bar = 50 μm). All data were shown as mean ± SD of three independent experiments (right, * P < 0.05, ** P < 0.01, Student’s t test). C Western Blot was used to detect the protein expressions of bcl-2, Bax and Cleaved-caspase-3, GAPDH was used as the internal control. D The mechanism scheme of RPL22L1 in GBM.

Article Snippet: Human GBM cell lines A-172 (CRL-1620 TM ), LN-229 (CRL-2611 TM ) and T98G (CRL-1690 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Negative Control, Apoptosis Assay, Flow Cytometry, TUNEL Assay, Western Blot, Control