Journal: Biomedicines

Article Title: Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells

doi: 10.3390/biomedicines8090296

Figure Lengend Snippet: Effect of ilimaquinone (IQ) on the viability of oral cancer cells (SCC4 and SCC2095). ( A ) SCC4 and ( B ) SCC2095. The cells were seeded onto 96-well plates, incubated for 24 (■) and 48 h (□), treated with IQ for 48 h and then cell viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Points, means; bars, S.D. ( n = 3–6). * p < 0.05 and ** p < 0.01 vs. the control group.

Article Snippet: SCC4 human oral squamous cell carcinoma cells were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan), and SCC2095 human oral squamous cell carcinoma cells were kindly provided by Professor Susan R. Mallery (The Ohio State University).

Techniques: Incubation, Control

Journal: Biomedicines

Article Title: Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells

doi: 10.3390/biomedicines8090296

Figure Lengend Snippet: Effect of ilimaquinone (IQ) treatment on apoptosis. ( A ) The percentage of apoptotic cells (Q2 + Q4) after dimethyl sulfoxide (DMSO) vehicle, IQ, or 50 nM staurosporine (Stauro.) treatment for 48 h. SCC4 cells were treated with either DMSO, IQ, or Stauro. in 5% fetal bovine serum (FBS)-supplemented DMEM/F12 medium for 48 h and stained with propidium iodide (PI)/annexin V. Columns, mean; bars, S.D. ( n = 4). ** p < 0.01 vs. the control group. ( B ) Concentration-dependent effect of IQ on caspase-3 activation. Staurosporine (Stauro.; 25 nM) was used as the positive control. Columns, mean; bars, S.D. ( n = 3). * p < 0.05 and ** p < 0.01 vs. the control group. ( C ) Expression of PARP, cleavage caspase-9, and procaspase-8 in SCC4 cells after 48 h treatment in 5% FBS-supplemented DMEM/F12 medium.

Article Snippet: SCC4 human oral squamous cell carcinoma cells were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan), and SCC2095 human oral squamous cell carcinoma cells were kindly provided by Professor Susan R. Mallery (The Ohio State University).

Techniques: Staining, Control, Concentration Assay, Activation Assay, Positive Control, Expressing

Journal: Biomedicines

Article Title: Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells

doi: 10.3390/biomedicines8090296

Figure Lengend Snippet: Effect of IQ on various biomarkers in SCC4 cells. ( A ) Phosphorylation/expression of Akt, p38, Bax, Mcl-1, Bcl-2, and survivin in SCC4 cells. Cells were treated with IQ for 48 h, and cell lysates were immunoblotted as described in Materials and Methods. ( B ) Expression of p-p53 and p53 of IQ in SCC4 cells. ( C ) Western blotting of p-p53 and p53 in SCC4 cells transiently transfected with a control vector or p53 shRNA for 24 h. ( D ) IQ-induced cell viability in p53 knockdown SCC4 cells. After incubation, cells were analyzed using the MTT assays. Columns, mean; bars, S.D. ** p < 0.01.

Article Snippet: SCC4 human oral squamous cell carcinoma cells were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan), and SCC2095 human oral squamous cell carcinoma cells were kindly provided by Professor Susan R. Mallery (The Ohio State University).

Techniques: Phospho-proteomics, Expressing, Western Blot, Transfection, Control, Plasmid Preparation, shRNA, Knockdown, Incubation

Journal: Biomedicines

Article Title: Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells

doi: 10.3390/biomedicines8090296

Figure Lengend Snippet: IQ increases reactive oxygen species (ROS) production. ( A ) Statistical analysis of ROS generation in SCC4 cells after the treatment with only 5 μM of IQ for 3 h or in combination with 5 mM of N -acetylcysteine (NAC) or 5 mM of glutathione (GSH). Cells were stained with carboxy-DCF-DA and analyzed using flow cytometry. Data are expressed as mean ± S.D. ( n = 3). * p < 0.05 and ** p < 0.01. ( B ) IQ-induced H2AX phosphorylation/expression in SCC4 cells.

Article Snippet: SCC4 human oral squamous cell carcinoma cells were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan), and SCC2095 human oral squamous cell carcinoma cells were kindly provided by Professor Susan R. Mallery (The Ohio State University).

Techniques: Staining, Flow Cytometry, Phospho-proteomics, Expressing

Journal: Biomedicines

Article Title: Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells

doi: 10.3390/biomedicines8090296

Figure Lengend Snippet: Ilimaquinone (IQ) induces autophagy. ( A ) Left panel, SCC4 cells were treated with DMSO vehicle, IQ, or rapamycin (RAP) for 24 h, harvested and stained with acridine orange, and examined under a fluorescence microscope; arrows, acidic vesicular organelles (AVOs). Right panel, statistical analysis of AVOs in IQ-treated SCC4 cells. Columns, mean; bars, S.D. ( n = 3). * p < 0.05 and ** p < 0.01 vs. the control group. ( B ) LC3B-II and Atg5 expression in SCC4 cells after IQ treatment for 48 h. ( C ) SCC4 cells were pretreated 20 μM of 3-methyladenine (3-MA) or 20 μM of chloroquine (CQ) for 15 min, followed by incubation with 10 μM IQ for 48 h and dual staining with propidium iodide (PI)/annexin V- fluorescein isothiocyanate (FITC). Percentages in the graphs are representative of cell percentage in the respective quadrants ( n = 3). Columns, means; bars, S.D. * p < 0.05 and ** p < 0.01.

Article Snippet: SCC4 human oral squamous cell carcinoma cells were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan), and SCC2095 human oral squamous cell carcinoma cells were kindly provided by Professor Susan R. Mallery (The Ohio State University).

Techniques: Staining, Fluorescence, Microscopy, Control, Expressing, Incubation

Journal: Cell & Bioscience

Article Title: Histone methyltransferase KMT2D cooperates with MEF2A to promote the stem-like properties of oral squamous cell carcinoma

doi: 10.1186/s13578-022-00785-8

Figure Lengend Snippet: Expression pattern of KMT2D in patients with OSCC. A Representative immunohistochemical staining of KMT2D in normal mucosa and primary OSCC tissue of KMT2D(+) and KMT2D(−). (scale bars = 100 μm). B Quantification analysis of immunohistochemical histoscore of KMT2D among normal mucosa and OSCC. C KMT2D expression levels in OSCC tissues from different pathological grades. D KMT2D expression levels in OSCC tissues from different tumor sizes. E KMT2D mRNA expression levels of paired normal mucosa and OSCC from TCGA datasets. F KMT2D protein expression levels in different cell lines. G KMT2D protein expression in human OSCC tissues and adjacent normal mucosa from the same patient. Results are representative of at least three independent experiments (*p < 0.05, **p < 0.01). Data are presented as means ± SD. OSCC, oral squamous cell carcinoma; SD standard deviation, ns no significant, KMT2D(+) KMT2D positive, KMT2D(−) KMT2D negative

Article Snippet: The OSCC cell lines CAL27, SCC25 and SCC4 were obtained from the China Center for Type Culture Collection (Shanghai, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Standard Deviation

Journal: Cell & Bioscience

Article Title: Histone methyltransferase KMT2D cooperates with MEF2A to promote the stem-like properties of oral squamous cell carcinoma

doi: 10.1186/s13578-022-00785-8

Figure Lengend Snippet: KMT2D contributes to the self-renewal ability of primary OSCC cells. A Representative images showed the immunofluorescence staining of KMT2D in patient-derived organoids generated from OSCC and paracancerous specimens, as well as their parental tissues. (scale bars = 50 μm). B Representative images indicated the organoid reconstitution assay (i.e. organoid morphology and clone size) conducted for OSCC cells transfected with Sh-CON and Sh-KMT2D. C Quantificational analysis showed the organoid reconstitution assay (i.e. organoid forming efficiency) conducted for OSCC cells transfected with Sh-KMT2D and Sh-CON. Student's t-test. D Representative images showed the immunofluorescence staining of β-catenin and CD133 in patient-derived OSCC organoids transfected with Sh-CON and Sh-KMT2D. (scale bars = 50 μm) Results are representative of at least three independent experiments(*p < 0.05,**p < 0.01, and ***p < 0.001). Data are presented as means ± SD. OSCC oral squamous cell carcinoma, SD standard deviation

Article Snippet: The OSCC cell lines CAL27, SCC25 and SCC4 were obtained from the China Center for Type Culture Collection (Shanghai, China).

Techniques: Immunofluorescence, Staining, Derivative Assay, Generated, Reconstitution Assay, Transfection, Standard Deviation

Journal: Cell & Bioscience

Article Title: Histone methyltransferase KMT2D cooperates with MEF2A to promote the stem-like properties of oral squamous cell carcinoma

doi: 10.1186/s13578-022-00785-8

Figure Lengend Snippet: Expression of KMT2D aggravates malignant behaviors of OSCC cells. A Immunoblotting of KMT2D in SCC4, SCC25, and CAL27 cells respectively transfected with Sh-CON and Sh-KMT2D. B Colony formation assays and quantification analysis generated from SCC4 and SCC25 cells transfected with Sh-CON and Sh-KMT2D. C Sphere formation assays and quantification analysis carried out from SCC4, SCC25, and CAL27 cells transfected with Sh-CON and Sh-KMT2D. (scale bars = 50 μm). D Wound healing assays and quantification analysis generated from SCC4, SCC25, and CAL27 cells transfected with Sh-CON and Sh-KMT2D. (scale bars = 50 μm). E Invasion assays and quantification analysis generated from SCC4, SCC25 cells transfected with Sh-CON and Sh-KMT2D. (scale bars = 50 μm). Results are representative of at least three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001). Data are presented as means ± SD; SD standard deviation

Article Snippet: The OSCC cell lines CAL27, SCC25 and SCC4 were obtained from the China Center for Type Culture Collection (Shanghai, China).

Techniques: Expressing, Western Blot, Transfection, Generated, Standard Deviation

Journal: Cell & Bioscience

Article Title: Histone methyltransferase KMT2D cooperates with MEF2A to promote the stem-like properties of oral squamous cell carcinoma

doi: 10.1186/s13578-022-00785-8

Figure Lengend Snippet: Knockdown of KMT2D impairs OSCC tumor growth in vivo . A Images of BALB/c nude mice from the two groups. B Images of xenograft tumors excised from BALB/c nude mice. C Quantification analysis of volumes of the xenograft tumors measured every 7 days on BALB/c nude mice. n = 8. D Weights of xenograft tumors excised from the BALB/c nude mice were evaluated. n = 8. E Representative hematoxylin and eosin (HE) and immunohistochemical staining of KMT2D and CSC-related markers in xenograft tumors. (scale bars = 50 μm). Results are representative of at least three independent experiments (*p < 0.05). CS cancer stem cell

Article Snippet: The OSCC cell lines CAL27, SCC25 and SCC4 were obtained from the China Center for Type Culture Collection (Shanghai, China).

Techniques: Knockdown, In Vivo, Immunohistochemical staining, Staining

Journal: iScience

Article Title: RUVBL1 accelerates tongue squamous cell carcinoma by mediating CRaf/MEK/ERK pathway

doi: 10.1016/j.isci.2024.109434

Figure Lengend Snippet:

Article Snippet: CAL-27, HUVEC, and SCC-4 cells were purchased from Wuhan Procell Life Technology CO., LTD. and Nanjing Cobioer Biosciences CO., LTD. Cultures of SCC-4 and CAL-27 cells were performed using DMEM medium (Thermo Fisher, USA).

Techniques: Virus, Recombinant, CCK-8 Assay, Staining, shRNA, Software, Flow Cytometry, Light Microscopy, Spectrophotometry, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: RUVBL1 accelerates tongue squamous cell carcinoma by mediating CRaf/MEK/ERK pathway

doi: 10.1016/j.isci.2024.109434

Figure Lengend Snippet:

Article Snippet: For the wound healing assay, 100 μl of SCC-4 and CAL-27 cells (2.5X10 4 cells/ml) were sequentially inoculated into two wells of the Ibidi insert (Ibidi, Germany).

Techniques: Virus, Recombinant, CCK-8 Assay, Staining, shRNA, Software, Flow Cytometry, Light Microscopy, Spectrophotometry, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: SCC4 and PCI-13 cells were treated for 2 days with 15 μM cisplatin. ALDH activity was determined using an Aldefluor assay in surviving cells and compared with the non-treated cells to evaluate the effect of cisplatin. The fluorescence intensity (ALDH activity) was determined by flow cytometry. Results represent the means and SEMs of two independent experiments. (* and # p < 0.05 compared to respective controls, t -test).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: Activity Assay, Fluorescence, Flow Cytometry

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: ( A ) Western blot analyses of human primary tumor homogenates from HNSCC patients using GAPDH as a loading control. ( B ) SCC4 and PCI-13 cells were treated with cisplatin (15 μM) for 2 and 4 days and total cell lysates were analyzed by Western blot for ALDH3A1 protein. Densitometric analysis of ALDH bands obtained by Western blot using Image J software shows relative levels after normalization for equal protein loading using β-tubulin as a loading control. Results are expressed as mean±SEM. (* p < 0.05 vs . respective control, t -test).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: Western Blot, Control, Software

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: ( A ) Structure of Alda-89, a small molecule ALDH3A1 activator, is shown (4-Allyl-1,2-methylenedioxybenzene, MW 162.1). ( B – C ) SCC4 cells and PCI-13 cells were treated with increasing concentrations of Alda-89 (15–60 μM) and/or cisplatin (15 μM) for four consecutive days. Then, the cells were analyzed on the fourth day. The percentage of live cells is shown compared to that of control cells. Cell viability was quantified using MTT assay, which was performed in 4–8 replicates in two independent experiments. Results represent mean ± SEMs (* p < 0.05 vs . respective cisplatin-only controls ( t -test)).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: Control, MTT Assay

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: ( A ) ALDH3A1 activity in SCC4 xenograft tumor. Mice with SCC4 xenografts were treated intra-tumorally with Aldi-6 or vehicle control for three consecutive days, and the ALDH3A1 activity was measured using an isoelectric focusing assay (see Methods). Experiment was performed three times. ( B ) Representative FACS analyses of ALDH activity of SCC4 and PCI-13 cells. After a two-day treatment of cisplatin (0.88 μM) and/or Aldi-6 (30 μM), ALDH activity was measured in the surviving cells by Aldefluor assay. Grey line represents the DEAB-treated negative control for each treatment condition. Blue line represents the ALDH activity of each sample. ( C ) Changes in ALDH activity in (B) were quantified as a ratio of the shift of MFI between treated and untreated sample. The ratio in MFI shift was calculated by (MFI of treated sample-(MFI of treated sample+DEAB))/(MFI of untreated sample-(MFI of untreated sample+DEAB)). Results represent the means ± SEMs of 2–3 independent experiments with 10,000 cells each. (* p < 0.05 vs . untreated control and ** p < 0.05 vs . cisplatin control ( t -test)).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: Activity Assay, Control, Negative Control

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: ( A ) Knockdown of ALDH3A1 by lentiviral transduction of shRNA in SCC4 cells was confirmed by Western blot assay. ( B ) Wild type (WT) and ALDH3A1 knockdown cells were treated with cisplatin on days 1 and 2, and cell viability was quantified by MTT on the fourth day. Results were expressed as percent of control (* p < 0.05 vs . cisplatin treated control, t -test).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: Knockdown, Transduction, shRNA, Western Blot, Control

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: ( A ) SCC4 and ( B ) PCI-13 cells were treated with Aldi-6 and/or cisplatin for two consecutive days. Then, the cells were analyzed on the fourth day. Cell viability was quantified using MTT assay. Results represent mean ± SEMs of 8–16 replicates. *p < 0.05 and ** p < 0.0001 vs . control; # p < 0.005 vs . cisplatin only group; ## p < 0.05 vs . Aldi-6 only group ( t -test).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: MTT Assay, Control

Journal: Oncotarget

Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

doi: 10.18632/oncotarget.17017

Figure Lengend Snippet: ( A ) SCC4 cells (2 × 10 6 ) were subcutaneously injected into the flanks of NOD- scid IL2Rgamma null mice ( n = 3–6 per group). Mice were treated systemically with Aldi-6, using implantable osmotic mini pumps (24 mg/kg/day) for continuous delivery of the compound. Cisplatin was administered by weekly i.p. injection (2 mg/kg) for 3 weeks. Tumor size was measured weekly for three weeks. One-way ANOVA analysis was performed on the final tumor size (* p < 0.05). ( B ) Quantification of the final tumor volumes (* p < 0.05 vs . control; and # p < 0.05 vs . cisplatin, t -test, n = 3–6 per cohort).

Article Snippet: Two million SCC4 cells were injected subcutaneously in the flank in a mixture of 1:1 PBS and Matrigel (Becton Dickinson).

Techniques: Injection, Control