Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

Article Title: Antiferroptotic properties of allicin and related organosulfur compounds-diallyl disulfide and diallyl trisulfide-from Garlic.

doi: 10.1016/j.fct.2024.115124

Figure Lengend Snippet: Fig. 4. The antiferroptotic effect of allicin is independent of Nrf2-regulated phase II enzyme expression. A. Western blot analysis showing that allicin upregulated expression of the phase II enzymes GCLM, GCLC, and HO-1 in WT HT22 cells but not Nrf2-KD HT22 cells. Cells were incubated with allicin (1 or 10 μM) for 16 h (Upper) Representative immunoblot. (Lower) Band intensities as quantified using ImageJ. Results are presented as the mean ± SD. *P < 0.05; ****P < 0.0001; ns, not significant. B. Allicin protected both WT and Nrf2-KD HT22 cells against erastin-induced death. Cells were treated with the indicated concentrations of allicin in the absence or presence of erastin for 24 h. Cell death was measured using an LDH assay. Results are presented as the mean ± SD. ###P < 0.001; ####P < 0.0001 compared to the corresponding erastin alone by ANOVA with post hoc Tukey’s test.

Article Snippet: Nrf2-knockdown (KD) HT22 cells were generated using the Nrf2 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc-421869) and the Nrf2 HDR plasmid (Santa Cruz Biotechnology, cat# sc-421869-HDR) as previously described (Hirata et al., 2020).

Techniques: Expressing, Western Blot, Incubation, Lactate Dehydrogenase Assay

Journal: International Journal of Biological Sciences

Article Title: TET1s deficiency exacerbates oscillatory shear flow-induced atherosclerosis

doi: 10.7150/ijbs.69281

Figure Lengend Snippet: CX40 mediates TET1s-induced endothelial barrier reinforcement. (A) Heatmap of the top 20 selected upregulated genes by RNA sequencing. (B) RT-qPCR was used to test the mRNA levels of the top 5 upregulated genes from RNA-seq and three hemodynamic-sensitive genes. (C) The CX40 protein expression level was quantified by WB (n=6 per group). (D-L) Stable CX40 -/- p-HUVECs were generated by transfecting human connexin 40-specific CRISPR/Cas9 KO plasmids. Then, TET1s-adenovirus was used to transfect CX40 -/- and CX40 +/+ p-HUVECs to generate CX40 +/+ +NC, CX40 +/+ +OE, CX40 -/- +NC and CX40 -/- +OE p-HUVECs. (D) The fluorescence intensity of the lower chamber medium was tested as described in Fig. C (n>6 per group). (E, H) Immunofluorescence staining for F-actin and VE-cadherin. The green dotted line indicates the intercellular space area. (F-G) Quantitative analysis of single-cell F-actin length and intercellular space area to image E (n>10 per group). (I-K) Quantitative analysis of VE-cadherin discontinuity, intercellular space area and ratio of VE-cadherin in several morphological categories to image H (n>10 per group). All data were presented as the mean ± SD.

Article Snippet: P-HUVECs were transfected at 60-70% confluence with connexin 40 (CX40) CRISPR/Cas9 KO plasmids (h) (sc-401031, Santa Cruz Biotechnology) and CX40 HDR (sc-401031-HDR, Santa Cruz Biotechnology) using UltraCruz® Transfection Reagent (sc-395739, Santa Cruz Biotechnology) according to the manufacturer's protocol.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Generated, CRISPR, Fluorescence, Immunofluorescence, Staining, Quantitative Single Cell

Journal: International Journal of Biological Sciences

Article Title: TET1s deficiency exacerbates oscillatory shear flow-induced atherosclerosis

doi: 10.7150/ijbs.69281

Figure Lengend Snippet: TET1s increases CX40 expression by inhibiting histone deacetylation on the promoter of CX40. (A-B, D-E) p-HUVECs were transfected with TET1s-overexpressing adenovirus and negative control adenovirus and further tested after 48 h. (A) The global protein levels of ac-H3K27 and H3K27 in p-HUVECs were tested by Western blot (n=6 per group). (B) Sin3a interaction with TET1s and TET1-FL was analyzed by Co-IP (n=3 per group). (C) Schematic of human CX40 promoter and CHIP-qPCR products. TS indicates transcriptional start; P1-P5 indicates primer 1-primer 5; F indicates forward primer, R indicates reversed primer. (D-E) ChIP-qPCR was used to test Sin3a and ac-H3K27 enrichment in the CX40 promoter (-550 bp to +43 bp) (n=4 per group). (F-G) p-HUVECs were transfected with TET1s-overexpressing adenovirus and negative control adenovirus for 48 h and added HATI2 to media. (F) ChIP-qPCR was used to test ac-H3K27 enrichment in the CX40 promoter. (G) The CX40 mRNA levels were tested by RT-qPCR (n=4 per group). All data were shown as the mean ± SD.

Article Snippet: P-HUVECs were transfected at 60-70% confluence with connexin 40 (CX40) CRISPR/Cas9 KO plasmids (h) (sc-401031, Santa Cruz Biotechnology) and CX40 HDR (sc-401031-HDR, Santa Cruz Biotechnology) using UltraCruz® Transfection Reagent (sc-395739, Santa Cruz Biotechnology) according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Negative Control, Western Blot, Co-Immunoprecipitation Assay, ChIP-qPCR, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: ( A – C ) Fhit overexpression activates caspase-dependent apoptotic pathway in NSCLC cells. H460 cells were transduced with Ad-Fhit (A). Two days after transduction, apoptosis was examined by trypan blue staining (B) and Western blot analysis (C). LacZ was used as a negative control. MOI, multiplicity of infection; NT, not treated. *** p < 0.001. ( D ) Serum starvation induces autophagy and Fhit is up-regulated during this process. HCC827 and Calu-3 cells were kept in normal culture conditions (10% FBS, +) or serum starved (−) and then cell lysates were analyzed by Western blotting with specific antibodies. ( E ) The effect of Fhit knockout on autophagy induced by serum deprivation. Endogenous Fhit was knocked out using a CRISPR/Cas9 knockout (KO) plasmid and autophagy marker proteins were analyzed by Western blotting after 24 h of serum deprivation in HCC827 cells. wt , wild type; ko , knockout.

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: Over Expression, Transduction, Staining, Western Blot, Negative Control, Infection, Knock-Out, CRISPR, Plasmid Preparation, Marker

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: ( A ) Effect of Ad-Fhit transduction on morphology of H460 cells as shown by light microscopy (magnification ×400). ( B ) Effect of Fhit gene transduction on expression of autophagy marker proteins in Fhit-deficient NSCLC cells. Autophagy marker proteins were assessed by Western blot analysis 48 h after infection. Ad-LacZ-transduced cells were used as a nonspecific control for adenoviral vector-mediated gene transfer. MOI, multiplicity of infection. ( C ) Assessment of autophagy with immunofluorescence. Fhit and p62 were co-immunostained 48 h after infection with Ad-Fhit in H460 cells (left panel). Nuclei were stained with Hoechst 33342 (blue). Expression of Fhit protein is shown in green, and expression of p62 protein is shown in red. Arrowheads, cells infected with Ad-Fhit. The number of p62 puncta was determined (right panel). Results are presented as mean ± standard deviation of four independent experiments. *** p < 0.001. ( D ) Acridine orange staining 48 h after infection with Ad-Fhit or Ad-LacZ into H460 cells. Acidic vesicular organelles (AVO) were analyzed by confocal fluorescent microscopy (×400, top) and FACS analysis (bottom). NT, not treated.

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: Transduction, Light Microscopy, Expressing, Marker, Western Blot, Infection, Control, Plasmid Preparation, Immunofluorescence, Staining, Standard Deviation, Microscopy

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: Top 20 increased or decreased proteins upon transduction of the Fhit gene

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: Transduction, Binding Assay

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: ( A ) Antibody arrays of cell lysates from H460 cells infected with Ad-Fhit or Ad-LacZ. Numbers in parentheses represent signal intensity. ( B ) Expression of 14-3-3 isoforms was assessed by Western blot analysis in H460 cells infected with Ad-Fhit or Ad-LacZ. MOI, multiplicity of infection. ( C ) Immunofluorescence images of Fhit and 14-3-3τ expression in H460 cells infected with Ad-Fhit or Ad-LacZ (upper panel). Nuclei were stained with Hoechst 33342 (blue). Expression of Fhit protein is shown in red, and expression of 14-3-3τ protein is shown in green. The intensity of 14-3-3τ staining per cell was assessed (lower panel). Results are presented as mean percentage ± standard deviation ( n = 3). * p < 0.05. ( D ) Immunoprecipitation analysis for the interaction of Fhit and 14-3-3τ proteins in NSCLC cells. Cell extracts were prepared from H460 cells transduced with Ad-Fhit, and immunoprecipitations (IP) were carried out by incubating the cell lysates with anti-FLAG antibody followed by immunoblotting with 14-3-3 antibodies, and vice versa.

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: Infection, Expressing, Western Blot, Immunofluorescence, Staining, Standard Deviation, Immunoprecipitation, Transduction

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: ( A ) Immunofluorescence images of Fhit and beclin-1 expression in H460 cells infected with Ad-Fhit or Ad-LacZ (upper panel). Nuclei were stained with Hoechst 33342 (blue). Expression of Fhit protein is shown in green, and expression of beclin-1 protein is shown in red. The intensity of beclin-1 staining per cell was assessed (lower panel). Results are presented as mean percentage ± standard deviation ( n = 5). *** p < 0.001. ( B ) Effect of 14-3-3τ silencing on expression of autophagy marker proteins. H460 cells were pretreated with 14-3-3τ siRNA (si14-3-3τ) for 24 h and then transduced with Ad-Fhit or Ad-LacZ for 48 h. Autophagy marker proteins were assessed by Western blot analysis. Scrambled siRNA was used as a nonspecific siRNA control. ( C ) Acridine orange staining in Ad-Fhit- or Ad-LacZ-infected H460 cells with or without pre-treatment with siRNA against 14-3-3τ (si14-3-3τ). Acidic vesicular organelles (AVO) were analyzed by confocal fluorescent microscopy (×200) (left panel). The number of AVO per cell was assessed (right panel). Data are presented as mean ± standard deviation of six independent experiments. *** p < 0.001 and ††† p < 0.001. siControl, contol siRNA.

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: Immunofluorescence, Expressing, Infection, Staining, Standard Deviation, Marker, Transduction, Western Blot, Control, Microscopy

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: ( A ) Effects of autophagy inhibition on viability of H460 cells transduced with Ad-Fhit. H460 cells, stably expressing shRNA against beclin-1 (shBeclin-1) or LC3B (shLC3) to block autophagy, were transduced with Ad-Fhit and cell viability was analyzed by a WST assay. Results are mean percentage ± standard deviation of three independent experiments. †† p < 0.01 and ††† p < 0.001 for the Fhit transduction with beclin-1 or LC3 shRNA treatment versus the Fhit transduction with control shRNA (shControl) treatment. MOI, multiplicity of infection. ( B ) Western blot analysis of apoptotic protein expression after Fhit transduction in H460 cells stably expressing shRNA against beclin-1 (shBeclin-1) or LC3B (shLC3). shControl, control shRNA. ( C ) Effects of 14-3-3τ inhibition on viability of H460 cells transduced with Ad-Fhit. H460 cells were transfected with siRNA against 14-3-3τ (si14-3-3τ) and then transduced with Ad-Fhit. Cell viability was analyzed by a WST assay. Scrambled siRNA (siControl) was used as a nonspecific siRNA control. Results are mean percentage ± standard deviation of three independent experiments. ††† p < 0.001 for the Fhit transduction with 14-3-3τ siRNA treatment versus the Fhit transduction with control siRNA treatment. MOI, multiplicity of infection. ( D ) Western blot analysis of apoptotic protein expression after Fhit transduction in H460 cells that had been transfected with siRNA against 14-3-3τ (si14-3-3τ). siControl, control siRNA. ( E ) Effects of pharmacological inhibition of autophagy by hydroxychloroquine (HCQ) on the viability of H460 cells transduced with Ad-Fhit. H460 cells were transduced with Ad-Fhit and then treated with 10 μM of HCQ or PBS (vehicle) as a control. Cell viability was assessed by a WST assay. Results are presented as mean percentage ± standard deviation of three independent experiments. ††† p < 0.001 for the Fhit transduction with HCQ treatment versus the Fhit transduction with vehicle treatment. ( F ) Western blot analysis of apoptotic protein expression in H460 cells that were transduced with Ad-Fhit and treated with 10 μM of HCQ.

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: Inhibition, Transduction, Stable Transfection, Expressing, shRNA, Blocking Assay, WST Assay, Standard Deviation, Control, Infection, Western Blot, Transfection

Journal: Oncotarget

Article Title: Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells

doi: 10.18632/oncotarget.16652

Figure Lengend Snippet: in vivo . ( A ) Effects on tumor growth. H460 cells, stably expressing shRNA against beclin-1 (shBeclin-1) or LC3B (shLC3), were xenografted in nude mice. After tumors reached a volume of 100 mm 3 , a lentiviral vector expressing the Fhit gene (Fhit) or LacZ (LacZ) was injected into the tumors. Tumor volumes were measured daily. Results are reported as the mean ± standard deviation from six mice in each group. *** p < 0.001 for the control shRNA (shControl) + LacZ group versus the shControl + Fhit group and for the shControl + Fhit group versus the shBeclin-1 or shLC3 + Fhit group. ( B ) Tumors formed in nude mice. ( C ) Expression of apoptotic and autophagic proteins in tumors. Crude protein lysates were prepared from tumor samples on day 11 after injection and analyzed by Western blotting. ( D–F ) Effects of pharmacological inhibition of autophagy on the antitumor effect of Fhit protein. Hydroxychloroquine (HCQ, 60 mg/kg of body weight) or PBS was injected into the peritoneal cavity daily after intratumoral injection of lentiviral-Fhit (Fhit) or lentiviral-LacZ (LacZ). Results are shown as the mean ± standard deviation from five mice in each group. ** p < 0.01 for the Fhit + PBS group versus Fhit + HCQ group.

Article Snippet: HCC827 (Fhit-positive) NSCLC cells were transfected with the Fhit CRISPR/Cas9 KO plasmid and Fhit homology-directed repair (HDR) plasmid (Santa Cruz Biotechnology) containing a puromycin resistance gene for the selection of cells with DNA double strand breaks (DSB) in the Fhit gene.

Techniques: In Vivo, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Injection, Standard Deviation, Control, Western Blot, Inhibition

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 3. RNA sequencing reveals WNT2 up-regulation upon sotorasib treatment. Volcano plot representing gene expression changes between (A) H23 parental cells, sotorasib treated versus untreated, and (B) isogenic resistant H23 cells compared to parental cells. Statistical significance was calculated for control verse treatment. n = 3 per group. (C) Number of overlapping and unique genes that were up-regulated or down-regulated with respect to treatment represented as a Venn diagram. (D) Heatmap representing top 10 overlapping up-regulated and down-regulated genes based on average fold change. (E and F) Effect of 3.2 μM sotorasib on H23 parental or isogenic resistant (Iso R) cells having knockdown of ITGB4 or CTNNB1 or both represented as percent change in growth at 96 hours (bar graph), respectively. Two-way ANOVA was used to calculate the statistical significance for each time point and for each condition (si Control, si ITGB4, si CTNNB1, si CTNNB1 + si ITGB4; n = 3 per group; ****P < 0.0001. (G) Immunoblot confirmed knockdown of ITGB4 and β-catenin in H23 parental cells and H23 sotorasib (20 μM) resistant cells. These cells were also treated with 3.2 μM sotorasib for 72 hours to identify changes in protein expression and signaling. (H) Representing the effect of ITGB4 and CTNNB1 single knockdown or double knockdown together with 10 μM sotorasib as a percent change in growth. Two-way ANOVA was used to calculate the statistical significance for each time point and each condition (si Control, si PXN, si ITGB4, and si PXN + si ITGB4; n = 3 per group; ****P < 0.0001. (I) Immunoblot showing knockdown of ITGB4 and β-catenin and changes in expression of active β-catenin, γH2AX, and p27 in SW1573 cells. (J) Immunoblot showing the reduction in the expression of WNT2, ITGB4, phospho, and total β-catenin in the SW1573 treated with sotorasib and CFZ drug combination.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: RNA Sequencing, Gene Expression, Control, Knockdown, Western Blot, Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 5. KRAS G12C inhibitors have a differential effect on cell growth and progression. (A) Effect of increasing concentrations (1 to 4 μM) of sotorasib and adagrasib exhibited different effects on cell proliferation over 24 hours in H23 cells. Two-way ANOVA was used for calculating statistical significance across various time points and for each drug concentration. n = 3. (B) Effect of KRAS inhibitors sotorasib or adagrasib increasing concentrations (1 to 8 μM) on SW1573 cells within 24 hours of drug treatment. Two-way ANOVA test was used to calculate the statistical significance. n = 3 sample per group. (C) Immunofluorescence image of SW1573 to support the dose dependent inhibitory effect of adagrasib on cell growth and expression of WNT2 (green) and phospsho-S675–β-catenin (magenta). The region of interest was zoomed 50% digitally to show membrane blebbing induced by adagrasib (white arrows). (D and E) Cell cycle dynamics were determined using IncuCyte Cell Cycle Lentivirus Reagent with fluorescence indicating cell cycle phase (brightfield image and schematic). Effect of increasing concentrations of sotorasib (1.25 to 20 μM) and adagrasib (0.6 to 1.25 μM) on cell cycle in SW1573 cells represented as pseudo color plots. The y axis of the plot represents events positive for GFP, and x axis represents the events positive for mKate2. mKate positive represents G1; GFP positive represents S, G2, and M; and double positive represents G1-S–transitioning cells.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: Concentration Assay, Immunofluorescence, Expressing, Membrane, Fluorescence

Journal: Neuron

Article Title: Sensory Discrimination of Blood and Floral Nectar by Aedes aegypti Mosquitoes

doi: 10.1016/j.neuron.2020.09.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: See the “ Transcript abundance and differential expression analysis ” section below for additional annotation information. . Ir7f-T2A-QF2 -SV40-3xP3-dsRed (Addgene plasmid#140942) 1 1 1 Plasmid backbone from pUC19 (Primers: Forward, 5’-attttgaggcgggCTAGAGTCGACCTGCAGGC-3’; Reverse, 5’-aatcagccagtcaCCCGGGTACCGAGCTCGA-3’).

Techniques: Sequencing, Recombinant, Plasmid Preparation, Software, Imaging