Journal: Advanced Science

Article Title: Engineered Bacteria‐Vesicle Delivered Lactate Reprogramming Boosts Tumor Radiosensitivity

doi: 10.1002/advs.202524303

Figure Lengend Snippet: In vitro cytotoxicity and mechanistic analysis of LOx‐loaded OMVs. (a) TEM characterization of OMVs‐P (OMVs derived from ENHP) and OMVs‐L (OMVs derived from ENHL) morphology; scale bar: 200 nm. (b,c) Flow cytometric analysis of CT26 cell apoptosis: representative plots (b) and quantitative analysis (c) following treatments (G1: PBS control; G2: OMVs‐P; G3: OMVs‐L; G4: 6 Gy; G5: OMVs‐P + 6 Gy; G6: OMVs‐L + 6 Gy; n = 5). (d) Cell viability assessment of CT26 cells post‐treatment ( n = 5). (e) Quantification of colony counts with different treatments towards CT26 cells. (f) Immunofluorescence images of γ‐H2AX staining after different treatments; scale bar: 100 µm. (g) The lactate content in the supernatant of CT26 cells after different treatments ( n = 5). (h) Western blot analysis of CT26 cell lysates for Pan‐Kla, GAPDH, and MRE11 expression, along with Pan‐Kla‐modified MRE11 levels under different treatments. All data represent mean ± SD. Statistical significance was determined by one‐way ANOVA with Tukey's multiple‐comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns. not significant).

Article Snippet: Primary antibodies included anti‐Syndecan‐1 antibody (Abcam, ab128936), anti‐Dnak antibody (Abcam, ab69617), Anti‐L‐Lactyl Lysine Rabbit mAb (PTM Bio, 9H1L6), Ki‐67 Polyclonal antibody (Proteintech, 28074‐1‐AP), HIF1α Rabbit pAb (ABclonal, A11945), LDHA Rabbit pAb (ABclonal, A1146), β‐Actin Rabbit mAb (ABclonal, AC050), DDDDK‐Tag Rabbit mAb (ABclonal, AE092), Myc‐Tag Rabbit mAb (ABclonal, AE070), GAPDH Rabbit mAb (ABclonal, A19056), MRE11 (Boster, BM5057), and Anti‐gamma H2A.X (Abcam, ab81299).

Techniques: In Vitro, Derivative Assay, Control, Immunofluorescence, Staining, Western Blot, Expressing, Modification

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 2 TINCR is involved in cell adhesion, cell migration, extrasellar matrix production, wound healing, tissue development and cell differentiation in human bronchial epithelial cells. (a) Venn diagram showing genes that are differentially expressed (>2.0 or <0.5 fold change) in NHBECs transfected with siTINCR1 or siTINCR2 compared to NHBECs transfected with siSCR. (b) Heat map representing color-coded expression levels of differentially expressed 603 genes in NHBECs transfected with siTINCR1 or siTINCR2 compared to NHBECs transfected with siSCR. Increased genes are presented on the heat map in yellow and decreased genes in purple (c) Bar plots of the p value for GO term enrichment (GO Process) for differentially expressed genes. (d) qRT-PCR data; and (e) Western blot analysis in NHBECs transfected with siSCR, siTINCR1 or siTICNR2. *p < 0.05. NHBECs were cultured in 6 well plate for 4 days after transfection. Data presented are from one of two independent experiments with similar results. (f-g) qRT–PCR and western blot analysis of TINCR, TP63, COL1A1, FN1, IL-6, and IL1β expression for NHBECs transfected with pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR. *p < 0.05. Data presented are from one of two independent experiments with similar results. (h) Representative image of scratch cell migration assay for NHBECs transfected with siSCR or siTINCR at 0 and 24 h. The red solid lines define the areas lacking cells. The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch toward the center of the scratch. Ten randomly selected fields were measured for the rate of migration and compared between siSCR and siTINCR. *p < 0.05. (i) Cell proliferation was measured by WST-1 assay for NHBECs transfected with siSCR or siTINCR

Article Snippet: The cells were incubated with TP63 (Cat. No. V3815SAF, NSJ Bioreagents) and FOXJ1 (cat. No. 14-9965-80.

Techniques: Migration, Cell Differentiation, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Cell Migration Assay, WST-1 Assay

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 3 Loss of TINCR expression induced aberrant bronchial cell differentiation in ALI culture model and 3D organoid formation assay. (a) qRT–PCR analysis of relative TINCR, TP63, SCGB1A1, FOXJ1 and MUC5B expression (mean ± SEM) (b) Schematic overview of NHBECs differentiation with transfection of siRNA in ALI culture model. NHBECs were seeded in transwell inserts and expanded for 2–3 days until confluency was reached. NHBECs were transfected with siSCR or siTINCR at day-1, subsequently airlifted and basolateral media was exchanged to differentiation medium at day0. The medium was renewed every 2 days and samples were taken for analysis at 3, 7, and 14 days after airlift. (c) qRT–PCR analysis of TINCR, TP63, SCGB1A1, FOXJ1, MUC5B, COL1A1, FN1, IL-1β and IL-6 expression at day3 and day14 after airlift with siRNA. *p < 0.05. Data presented are from one of two independent experiments with similar results. (d) Immuofluorescence staining of TP63 and FOXJ1 for NHBEC transfected with siSCR and siTINCR day day3 and day14. Green represents TP63, and red represents FOXJ1 staining. Blue represents nuclear DNA staining by Hoechst33342; bars =20 μm. Quantitative analysis of the singal intensity per cell per group. Bars represent mean score ± SEM.; *p < 0.05. (e) 3D organoid formation assay for NHBECs transfected siSCR or siTINCR (day14). qRT–PCR analysis of relative gene expression of TINCR, TP63, SCGB1A1, FOXJ1 and MUC5B at day14. *p < 0.05. Data presented are from one of two independent experiments with similar results. (f) Representative image of bronchial cell organoid in NHBECs transfected siSCR or siTINCR; bars =100 μm. (g) Scatter dot plot of organoid size (left) and box plot of colony formation efficacy (right). *p < 0.001. Data presented are from one of two independent experiments with similar results. (h) qRT–PCR analysis of relative TINCR, TP63, SCGB1A1, FOXJ1 and MUC5B expression at day3 and day14 after airlift with transfection of the plasmids. *p < 0.05. Data presented are from one of two independent experiments with similar results

Article Snippet: The cells were incubated with TP63 (Cat. No. V3815SAF, NSJ Bioreagents) and FOXJ1 (cat. No. 14-9965-80.

Techniques: Expressing, Cell Differentiation, Tube Formation Assay, Quantitative RT-PCR, Transfection, Staining, Gene Expression

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 5 TINCR binds to STAU1 protein and controls critical regulators of differentiation. (a) Bar graphs show percentage of TINCR in the cytoplasm (black) and nucleus (white). NEAT1 serves as a positive control for nucleus enriched RNA and GAPDH serves as a positive control for cytoplasmic RNA. Data presented are from two independent experiments. (b) RIP experiments were performed using isotype IgG and STAU1 antibody to immunoprecipitated STAU1 protein/mRNAs complexes in total-cell extracts of NHBECs, and relative enrichment was determined as RNA associated with STAU1 IP relative to an input control. Relative ARF1 enrichment served as a positive control and NEAT1 as a negative control as NEAT1 does not interact with STAU1. Data presented are from two independent experiments. (c) Relative mRNA enrichment of STAU1 antibody in total-cell extracts of NHBECs transfected with siSCR or siTICNR. Data presented are from two independent experiments. (d and e) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU1. NHBECs were seeded on 6 well plate at 2 × 105 density and analyzed at day4 after transfection of siRNA reagents. *p < 0.05. (f and g) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU after transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR for 4 h. NHBECs were seeded on 24 well plate at 1 × 105 density and analyzed at day4 after transfection of siRNA reagents

Article Snippet: The cells were incubated with TP63 (Cat. No. V3815SAF, NSJ Bioreagents) and FOXJ1 (cat. No. 14-9965-80.

Techniques: Positive Control, Immunoprecipitation, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing