human-derived oscc (sas) cell lysates  (SAS institute)

Journal: Nucleic Acids Research

Article Title: Mechanism of Cas9 inhibition by AcrIIA11

doi: 10.1093/nar/gkaf318

Figure Lengend Snippet: AcrIIA11 inhibition mechanism. ( A ) Cas9 initially binds off-target DNA and samples PAMs (gray) in search of the PAM (black square) that is adjacent to the target site. Next, Cas9 forms an R-loop and cleaves the target DNA. ( B ) AcrIIA11 forms a 1:1 complex with Cas9. The complex binds PAM-rich off-target sites and prevents SaCas9 diffusion and target search. ( C ) AcrIIA11 also inhibits nuclease activity for those Cas9s that found the target.

Article Snippet: The TS–SUMO– Sa Cas9–sgRNA cell lysate was applied to a 5 ml of Strep-Tactin Superflow 50% suspension (IBA Life Sciences, 2-1206-025) gravity column equilibrated in Lysis Buffer containing 200 mM NaCl, 25 mM HEPES, pH 7.5, and 2 mM DTT supplemented with protease inhibitors and DNase.

Techniques: Inhibition, Diffusion-based Assay, Activity Assay

Journal: bioRxiv

Article Title: Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers

doi: 10.1101/2023.07.30.551184

Figure Lengend Snippet: a , Malignant lesion (white arrow) on the tongue of a patient having OSCC. b , Visualization of neoplastic lesions (white asterisks) using a handheld device reveals a loss of fluorescence in multiple areas (white letters), which may or may not correspond to definitive lesions; the handheld device is based on loss of free FAD autofluorescence.

Article Snippet: We assessed the spectral characteristics of non-cancer (HaCaT) and human-derived OSCC (Ca9–22, HSC-3 and SAS) cell lysates over a range of wavelengths that included those applied by optical devices.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers

doi: 10.1101/2023.07.30.551184

Figure Lengend Snippet: a , The fluorescence EEM of free FAD is characteristically bimodal, having two emission peaks at 530 nm with different (360 nm, 445 nm) excitation wavelengths. b , The EEM of reduced nicotinamide adenine dinucleotide (NADH) is unimodal (excitation at 360 nm, emission at 460 nm); the red dashed rectangles indicate the range of wavelengths (of free FAD) observable with handheld devices, and each contour corresponds to a region of equal fluorescence intensity. c-f , In comparison to non-cancer (HaCaT; c ) lysates, representative EEM spectra (quantified in ) suggest loss of intensity in OSCC cell lysates ( d-f ) but there are more non-fluorescent than fluorescent regions within the red dashed rectangles; red dots and triangles correspond to the peaks for FAD and NADH, respectively. g, Simulated representation of subtracting EEMs of HaCaT lysates from itself. h-j , Subtracting the EEMs of OSCC from the EEM of HaCaT cell lysates reveals that there are regions in the EEMs that are not within red dashed rectangles; additionally, there is a spectral region at excitation (360–400 nm) and emission (575–650 nm) that is distinct in all OSCC lysates.

Article Snippet: We assessed the spectral characteristics of non-cancer (HaCaT) and human-derived OSCC (Ca9–22, HSC-3 and SAS) cell lysates over a range of wavelengths that included those applied by optical devices.

Techniques: Fluorescence, Comparison

Journal: bioRxiv

Article Title: Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers

doi: 10.1101/2023.07.30.551184

Figure Lengend Snippet: a , Representative SDS-PAGE gel and quantitation when exposed to blue light suggest that decreased autofluorescence occurs at the 70 kDa region, and that fluorescence intensity is lower in OSCC cell (SAS, HSC-3 and Ca9–22) lysates compared to non-cancer (HaCaT) lysates. b , Representative western blot image of proteins transferred from SDS-PAGE gel shows SDHA is detected in the 70 kDa region where autofluorescence was previously observed; also, SDHA levels decrease in OSCC compared to HaCaT cells. c , Western blot image of proteins transferred from SDS-PAGE gel shows ACADV is also detected at the 70 kDa region where autofluorescence was previously observed; however, ACADV levels do not decrease in OSCC compared to HaCaT cells. d , GAPDH levels in western blots are similar between OSCC and HaCaT cells. Not significant (ns); Kruskal-Wallis test followed by Dunn’s multiple comparison test was used to assess ranks expressed as median (interquartile range) in f; Other datasets were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test and expressed as mean (SD); Grubb’s test eliminated an outlier in the HSC-3 group for ; n = 4–5 , n = 4 ( , f), n = 6 ( g).

Article Snippet: We assessed the spectral characteristics of non-cancer (HaCaT) and human-derived OSCC (Ca9–22, HSC-3 and SAS) cell lysates over a range of wavelengths that included those applied by optical devices.

Techniques: SDS Page, Quantitation Assay, Fluorescence, Western Blot, Comparison

Journal: bioRxiv

Article Title: Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers

doi: 10.1101/2023.07.30.551184

Figure Lengend Snippet: a , Representative phase contrast and immunofluorescent images reveal cytoplasmic localization of ACADV and SDHA in cell lines; scale bars are 200 μm. b , Relative fluorescence intensity of SDHA complexes recovered from cell lysates via immunoprecipitation with magnetic beads reveals decreased SDHA expression in OSCC (Ca9–22, SAS, HSC-3) when compared to non-cancer (HaCaT) cells.. One-way ANOVA followed by Tukey’s multiple comparison test, mean (SD), n = 3.

Article Snippet: We assessed the spectral characteristics of non-cancer (HaCaT) and human-derived OSCC (Ca9–22, HSC-3 and SAS) cell lysates over a range of wavelengths that included those applied by optical devices.

Techniques: Fluorescence, Immunoprecipitation, Magnetic Beads, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Parallel Reporter Assays Identify Altered Regulatory Role of rs684232 in Leading to Prostate Cancer Predisposition

doi: 10.3390/ijms22168792

Figure Lengend Snippet: SNP rs684232 alters FOXA1 binding. ( A ) Enrichment of rs684232 region in the FOXA1 ChIP DNA in 22Rv1 cells. IgG and Input as the negative control. Mean ± SD of three technical replicates. *** p < 0.001, two−tailed Student’s t -test. ( B ) Sanger sequencing chromatography of FOXA1 ChIP DNA for the rs684232 site in 22Rv1 cells. IgG and Input as the negative control. The position of rs684232 is highlighted with a yellow square and arrow. ( C ) Allele−specific enrichment of rs684232 site in the FOXA1 ChIP DNA determined by AS−qPCR in 22Rv1 cells. Mean ± SD of three technical replicates. ** p < 0.01, two−tailed Student’s t -test.

Article Snippet: Immunoprecipitation was performed with antibodies targeting FOXA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-22841, RRID:AB_2104862), H3K27ac (Abcam, Cambridge, UK, Cat# ab4729, RRID:AB_2118291), and H3K4me3 (Abcam, Cambridge, UK, Cat# ab8580, RRID:AB_306649).

Techniques: Binding Assay, Negative Control, Two Tailed Test, Sequencing, Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Parallel Reporter Assays Identify Altered Regulatory Role of rs684232 in Leading to Prostate Cancer Predisposition

doi: 10.3390/ijms22168792

Figure Lengend Snippet: rs684232 regulates gene expression of VPS53 , FAM57A , and GEMIN4 through FOXA1. ( A ) The location of the rs684232 relative to three target genes. ( B – D ) eQTL analysis in GTEx prostate tissues to reveal the association between alleles of rs684232 and VPS53 ( B ), FAM57A ( C ), and GEMIN4 ( D ) genes. p values are from a linear regression model. ( E ) Gene expression quantification of VPS53, FAM57A , and GEMIN4 by RT-qPCR in three 22Rv1 cells, including parental 22Rv1 cells and mutated cells 22Rv1(−/−) #1/ #2. Mean ± SEM of three biological replicates. ** p < 0.01, *** p < 0.001, two-tailed Student’s t -test. ( F ) Gene expression quantification of VPS53, FAM57A, and GEMIN4 by RT-qPCR in 22Rv1 cells treated with FOXA1 shRNA. Mean ± SD of three technical replicates. * p < 0.05, *** p < 0.001, two-tailed Student’s t -test. ( G – I ) Gene expression correlation analysis between transcription factor FOXA1 and the three target genes VPS53 ( G ), FAN57A ( H ), and GEMIN4 ( I ) in prostate tumor tissues from TCGA-PRAD database. Correlation coefficient ( r ) values and p values were from Pearson or Spearman correlation analysis, respectively. ( J – L ) Gene expression correlation analysis between FOXA1 and VPS53 ( J ), FAM57A ( K ), or GEMIN4 ( L ) in 33 kinds of cancer tissues from TCGA. The Pearson correlation coefficient value of each cancer type was plotted vs. the −log10 of p -value. The dot in yellow represents the PRAD. Correlation coefficient ( r ) values and p values were from the Pearson correlation analysis.

Article Snippet: Immunoprecipitation was performed with antibodies targeting FOXA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-22841, RRID:AB_2104862), H3K27ac (Abcam, Cambridge, UK, Cat# ab4729, RRID:AB_2118291), and H3K4me3 (Abcam, Cambridge, UK, Cat# ab8580, RRID:AB_306649).

Techniques: Gene Expression, Quantitative RT-PCR, Two Tailed Test, shRNA

Journal: Cells

Article Title: FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

doi: 10.3390/cells9091927

Figure Lengend Snippet: Pancreatic differentiation in presence of growth factors. ( a ) Gene expression of PDX1 , HLXB9 , HNF1B , HNF6 , SHH, TBX1, SOX9, AFP, and CDX2 measured by RT-qPCR in human embryonic stem cells (hESC)s at d0, d4, and d8 of differentiation treated ± 5 or 50 ng/mL fibroblast growth factor (FGF)2, FGF7, FGF10, or epidermal growth factor (EGF) in stage 2 medium. Values are means ± SEM, n = 4–6. 5/50 = 5 or 50 ng/mL growth factor, C = control medium without growth factors. ( b ) Fluorescence micrographs after pancreatic differentiation illustrating the protein expression of PDX1/HNF1B after a 4-day treatment ± 5 or 50 ng/mL FGF2, FGF7, FGF10, and EGF. Nuclei were counterstained with DAPI. Scale bar = 200 µm and 20 µm for higher magnification of the control. ( c ) Quantification of cell expansion using ± 50 ng/mL growth factor. Data are presented as mean (log2) ± SEM, n = 3. ( d ) Quantification of PDX1-positive cells upon treatment ± 5 or 50 ng/mL FGF2, FGF7, FGF10, and EGF at d8. Percentages are expressed as means ± SEM. *** = p ≤ 0.001, ** = p ≤ 0.01 compared to control, ANOVA plus Dunnett’s post-test.

Article Snippet: The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used.

Techniques: Gene Expression, Quantitative RT-PCR, Control, Fluorescence, Expressing

Journal: Cells

Article Title: FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

doi: 10.3390/cells9091927

Figure Lengend Snippet: Gene expression changes in response to different FGF2 concentrations. ( a ) hESCs were cultured in the presence of 5–50 ng/mL FGF2 during differentiation into PDX1-positive cells and compared to a control condition (=C). Gene expression changes of PDX1 , HNF1B , SHH, and TBX1 are presented as means ± SEM, n = 5–12. *** = p ≤ 0.001, ** = p ≤ 0.01, * = p ≤ 0.05 compared to control, ANOVA plus Dunnett’s post-test. ( b ) Effect of SHH inhibition by 10 µM Gant 58 or 2.5 µM Sant1 in stage 2 medium ± 50 ng/mL FGF2. Gene expression changes of PDX1 and SHH are presented as means ± SEM, n = 4–5. *** = p ≤ 0.001, compared to control, ANOVA plus Dunnett’s post-test.

Article Snippet: The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used.

Techniques: Gene Expression, Cell Culture, Control, Inhibition

Journal: Cells

Article Title: FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

doi: 10.3390/cells9091927

Figure Lengend Snippet: Inhibition of FGFR1c/3c rescues pancreatic development in presence of FGF2 and FGF7. ( a ) Gene expression changes of PDX1 , HNF1B , SHH , and TBX1 in d8 cells treated ± 10 ng/mL FGF2 (F2) and the FGFR1c/3c small molecule inhibitor PD-173074 (PD) with a concentration of 100 nM (PD). Data are means ± SEM, n = 4–5. *** = p ≤ 0.001, ** = p ≤ 0.01, * p = ≤0.05 ANOVA plus Tukey’s post-test, F2 treatment compared to F2 + PD and PD only. ( b ) Representative immunofluorescence staining of PDX1 in d8 control cells, cells treated with F2 and F2 + PD. Nuclei were counterstained with DAPI. Scale bar = 100 µm. ( c ) Gene expression changes of PDX1 , SOX9 , AFP , and CDX2 in d8 cells treated ± 50 ng/mL FGF7 (F7) and ± PD-173074 with a concentration of 10 or 100 nM (10, 100). Values are means ± SEM, n = 3–6, * = p ≤ 0.05, ANOVA plus Tukey’s post-test. ( d ) Quantification of PDX1-positive cells at d8 upon treatment ± 50 ng/mL FGF7 and ± PD-173074 with a concentration of 10 or 100 nM. Percentages are expressed as means ± SEM. * = p ≤ 0.05, ANOVA plus Tukey’s post-test.

Article Snippet: The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used.

Techniques: Inhibition, Gene Expression, Concentration Assay, Immunofluorescence, Staining, Control

Journal: Cell reports

Article Title: FOXA2 Is Required for Enhancer Priming during Pancreatic Differentiation

doi: 10.1016/j.celrep.2019.06.034

Figure Lengend Snippet:

Article Snippet: HNF1B , Santa Cruz , sc-22840x; RRID: AB_2279595.

Techniques: Recombinant, Transfection, Derivative Assay, Sequencing, CRISPR, Software

Journal: Cell reports

Article Title: FOXA2 Is Required for Enhancer Priming during Pancreatic Differentiation

doi: 10.1016/j.celrep.2019.06.034

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: rabbit anti-H3K4me1 (Abcam, ab8895, 5 μg), rabbit anti-H3K27ac (active motif, 39133, 5 μg), goat anti-FOXA2 (R&D, AF2400, 10 μg), rabbit anti-GATA6 (Cell Signaling Technology, 5851, 10 μg), goat anti-GATA4 (R&D, AF2606, 10 μg), rabbit anti-HNF1B (Santa Cruz, sc-22840x, 10 μg), goat anti-PDX1 (R&D, AF2419, 10 μg).

Techniques: Recombinant, Transfection, Derivative Assay, Sequencing, CRISPR, Software