Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Expression levels of ALDH1A1 and HLTF predict sensitivity to HCQ in cancer cell lines. (A) MTT (72 h) in colon and lung cancer cells. (B) Differentially expressed genes in HCQ-S (HT29) and HCQ-R (HCT15) colon cancer cells. (C) HCQ IC50 and Hill Slope for 33 human cancer cell lines. Blue dots indicate sensitive cell lines (<16 µM IC50); green indicates intermediate resistant cell lines (IC50 > 16 µM, Slope > −2.1); red indicates resistant cell lines (IC50 > 16, slope < − 2.1) (D) Protein expression detected by western blot of the 2 most upregulated (ALDH1A1, LYZ) and the 2 most downregulated (ABCB1, HLTF) genes in HCQ-sensitive (Sen), HCQ-intermediate resistant (Int Res), and HCQ-resistant (Res) cells. ANOVA indicates no single gene predicts sensitivity or resistance. (E) CART analysis of expression level of 4 genes (ALDH1A1, LYZ, HLTF, ABCB1) identifies a 2-gene signature that is sufficient to predict all sensitive cell lines. Exp.: expression as detected by fluorescence intensity of the band/ control.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Expressing, Western Blot, Fluorescence

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: ALDH1A1 levels control entry and activity of chloroquine derivatives in cancer cells. (A-B) Aldeflour assay shows (A) CQ derivatives produce no impairment of ALDH1 enzyme function, (B) siRNA against ALDH1A1 impairs enzymatic function. (C) DC341-C3 strucutre. (D) Fluorescence microscopy of DC340-Cy3 (red fluorescence) in A375 cells treated with nontargeting siRNA (NT) or siALDH1A1; or vehicle and DEAB for 24 h. Mean +/− SD from multiple experiments. (E) DC340-Cy3 fluorescence following SiNT or SiALDH1A1 in HT29 in the presence or absence of verapamil. (F) CD340-Cy3 in A375P cells transiently transfected with control or ALDH1A1-expressing vector. (G) Immunoblotting against autophagy markers in HCT15 cells transfected with control or ADLH1A1-expressing vector +- HCQ. Mean +/− SD for band quantification from multiple experiments shown. (H) LysoSensor fluorescence (green) of HCT15 and HT29 cells transfected with control or ALDH1A1-expressing vector, or siNT or siALDHA1, respectively. (I-K) 72-h MTT mean +/− SD. (I) Knockdown of ALDH1A1 promotes resistance to HCQ. (J) DEAB co-treatment promotes resistance to HCQ. (K) Overexpression of ALDH1A1 promotes HCQ-sensitivity. *p < 0.05.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Activity Assay, Fluorescence, Microscopy, Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Forced expression or knockdown of HLTF reverses HCQ sensitivity or resistance in cancer cells. (A) 2-wk colony formation assay, mean +/− SD. (B-C) immunoblotting and MTT assay of stable transfectants. (D) HCQ treatment +/- 5-azacytadine (aza). (E) Effects of knockdown of HLTF using shRNA on HCQ-R HCT15 cells. (F) Effects of overexpression or knockdown of HLTF on DC340-Cy3 uptake. (G) Sensitivity of HCQ and other lysosomal autophagy inhibitors depends on HLTF expression. HT29 vector (blue) and HT29HLTF (red) cells were treated as indicated. MTT at 72 h was measured. *p < 0.05.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Expressing, Colony Assay, Western Blot, MTT Assay, shRNA, Over Expression, Plasmid Preparation

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Overexpression of HLTF promotes resistance to Lys05 in HCQ-S tumors. (A) MTT (72 h) in HT29 vector and HT29 HLTF cells treated with the indicated combinations at 500 nM inhibitor +/− HCQ (10 µM). IGF1RAb, figitumumab; PTK2/FAKi, PF562271; PI3Ki, PF4691502. *P < 0.05. (B) HT29-vector and HT29-HLTF cells were grown as xenografts in nude mice. PBS or Lys05 (20 mg/kg) i.p. was adminstered daily. Daily tumor volumes mean +/− SEM. (C) Tumor volumes on the final day. (D) Tumor weights on the final day.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Over Expression, Plasmid Preparation

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: HCQ-associated ROS produces DNA damage that is either repaired by HLTF-POLH, or results in DSB. (A) Reactive oxygen species (ROS) measured by flow cytometry. (B) Phosphorylation of H2AFX. Band quantification presented as mean +/− SD for multiple experiments. (C) DNA fragmentation is more striking in HLTF− than HLTF+ cells. (D) Two-wk colony formation assay in HT29 cells with stable expression of vector or HLTF: 2 mM NAC rescues HCQ (10 µM) cytotoxicity. (E) Tiron rescues HCQ-associated DNA damage. (F) Immunofluorescence microscopy demonstrates HLTF abrogates HCQ-induced RAD52+ double strand breaks (arrow). Sale bar 100 µm (G) Knockdown of DNA POLH. Quantification of bands: mean +/− SD from multiple experiments; 72-h MTT with siNT compared to siPOLH abrogates HCQ resistance due to HLTF overexpression in HT29 cells. (H) ATM inhibition with KU-60019 mitigates HLTF-mediated rescue of HCQ cytotoxicity in 72-h MTT assay of HT29 vector and HT29HLTF cells. *p < 0.05; ns, not significant..

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Flow Cytometry, Colony Assay, Expressing, Plasmid Preparation, Immunofluorescence, Microscopy, Over Expression, Inhibition, MTT Assay

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Modulation of HCQ efficacy by ALDH1A1 and HLTF. (A) ROS levels in HCT15 and HT29 cells with overexpression or knockdown of ALHD1A1. (B) HCT15 cells transiently transfected with Control or ALDH1A1-expressing vector with or without HCQ (20 µM) for 12 h. (C) HT29 cells transfected with siNT (Non-Target) or siALDH1A1 with or without HCQ (20 µM) for 12 h. (D) Immunoblotting in lysates from the indicated cells treated with retinoic acid (RA 5 µM) +/− EZH2 inhibitor EPZ005687 2 µM (24 h). (E) Immunoblotting of lysates from HCT 15 cells transfected with control or ALDH1A1-expressing vector +/- EPZ005687 (2 µM), 24 h. (F) Immunoblotting from lysates from HCT15 cells treated with HCQ (20 µM) +/− Tiron (10 µM). (G) Immunoblotting of HCT15 cells transiently transfected with control or ALDH1A1-expressing vector +/− HCQ (20 µM) for 12 h. (H) ALDH1A1 facilitates entry of HCQ into the cell, where it accumulates in the lysosome. Lysosomal impairment leads to autophagy inhibition and the generation of ROS. ROS-associated DNA damage produces single-strand breaks with stalled replication forks. If HLTF is expressed, recruitment of the low fidelity DNA polymerase POLH allows translesion synthesis to occur promoting resistance to therapy. In the absence of HLTF-associated TLS, stalled replication forks collapse into double-strand breaks triggering cell death. Both RA and ROS can regulate KDM5D-PRC2-driven degradation of DNMT1 and upregulation of HLTF.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Inhibition, Translesion Synthesis

Journal: Archives of Toxicology

Article Title: The safety of nanostructured synthetic amorphous silica (SAS) as a food additive (E 551)

doi: 10.1007/s00204-016-1850-4

Figure Lengend Snippet: Genotoxicity of silica in vitro (including data of non-food-grade and colloidal SAS)

Article Snippet: HT-29 human colon carcinoma cell line , Pyrogenic SAS (AEROSIL ® 200, AEROSIL ® Ox50) , 12, 40 nm, 200, 50 m 2 /g , Evonik Industries , Cytotoxicity (±FCS, 1 and 10 %, 0.03–156.3 μg/cm 2 ); comet assay with and without Fpg , Negative, no oxidative DNA damage , Gehrke et al. ( ) .

Techniques: In Vitro, Mutagenesis, Spot Test, Derivative Assay, Single Cell Gel Electrophoresis, Incubation, Positive Control, Alkaline Single Cell Gel Electrophoresis

Journal: Cellular & Molecular Biology Letters

Article Title: Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium

doi: 10.1186/s11658-024-00537-1

Figure Lengend Snippet: Detection of CFTR protein by Western blot analysis in two pancreatic organoid lines (P) and the human intestinal cell line HT29-CL19A (I). Detection of e-cadherin (ECAD) served as a loading control. Outer left lane shows the position of molecular weight markers, with an approximate molecular mass as indicated

Article Snippet: The human colonic adenocarcinoma cell line HT29-CL19A (#T8212; Applied Biological Materials) was cultured in DMEM (Gibco), supplemented with fetal bovine serum (10%; Gibco), penicillin (100 U/mL; Gibco) and streptomycin (100 µg/mL; Gibco).

Techniques: Western Blot, Molecular Weight