Journal:

Article Title: Target Cell Range of Haemophilus ducreyi Hemolysin and Its Involvement in Invasion of Human Epithelial Cells

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: The bacterial strains and plasmids used in this study are described in Table and Fig. . Human cell lines were obtained from the American Type Culture Collection (Manassas, Va.). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Bacterial strain or plasmid Relevant characteristics Reference or source Strains E. coli HB101 Noninvasive strain, negative control 18 S. typhimurium SL1344 Highly invasive strain, positive control 16 H. influenzae Rd Nonhemolytic, noncytotoxic, negative control, ATCC 33391 M. Roberts, University of Washington H. ducreyi 35000 Wild type, hemolytic 41 35000ΔAPC 35000 hhdA :: cat , nonhemolytic, Cm r This study Plasmids pPT376 pUC19 with hhdA , Ap r 41 pBluescript KS(−) Cloning vector, Ap r Stratagene pBluescript SK(−) Cloning vector, Ap r Stratagene pPT376KS hhdA on pBluescript, Ap r This study pPT376ΔPst pPT376KS with internal hhdA Pst I fragment replaced with Bam HI linkers, Ap r This study pUC-ΔE cat Source of cat cassette, Cm r B.

Techniques: Plasmid Preparation, Negative Control, Positive Control, Cloning, TA Cloning

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 1. Dose- and time-dependent inhibition of bladder cancer cell migration by hAM preparations. (A–E′) Representative images of wound healing of T24 cells. (F–J′) Representative images of wound healing of RT4 cells. (K–O′) Representative images of wound healing of NPU cells. (P-R) Percentage of wound healing of T24, RT4 and NPU cells, respectively. Data are presented as mean ± standard error of the mean (SEM) of at least three independent experiments. *P < 0.05 vs. control group. Scale bars 50 µm.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Migration, Control

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 3. hAM homogenate and extract suppress the migration rate of non-invasive urothelial papilloma RT4 cells by decreasing the amount of lamellipodia and filopodia along the leading edge. RT4 cells treated with hAM homogenate, hAM extract or an appropriate culture medium (control) migrate collectively and maintain an epithelial morphology. However, migrating RT4 cells, treated with hAM preparations, contain less lamellipodia and filopodia (red arrows) along the leading edge than untreated control RT4 cells. Larger framed inset with black lines are the enlarged images of the corresponding smaller framed inserts. The presented images were acquired consecutively every 3 h and 15 min (shown in seconds) after the treatment. Shown is one representative experiment out of three independent experiments with different hAM preparations. Scale bars 50 µm.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Migration, Control

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 6. hAM homogenate and extract down-regulate FAK expression in bladder cancer urothelial cells but not in normal urothelial cells. (A) Western blot analysis of FAK and p-FAK in T24, RT4 and NPU cells treated for 24 h with hAM homogenate, hAM extract or appropriate culture medium (control samples) without hAM preparations. (A′) The expression and phosphorylation of FAK were normalised to the level of α-tubulin. In addition, the expression and phosphorylation of FAK in the treated samples were normalised to the control samples and these values are presented as log2 fold change (log2FC). The results are expressed as median (solid black line) with data range (minimum and maximum). The dashed black lines represent the normalised value of the control sample (0). The results were obtained on the basis of 3–6 biological replicates. The most representative blot of the mean effect is presented. The original blots are presented in Supplementary Fig. 2. The absence of full-length membranes is due to the cropping of the membranes prior to hybridization with primary antibodies. * p ≤ 0.05. (B) qPCR analysis of PTK2 expression in T24, RT4 and NPU cells treated for 24 h with hAM homogenate, hAM extract or appropriate culture medium without hAM preparations (control samples). PTK2 expression was normalised to GAPDH levels. The qPCR results were presented as ΔCt (CtGAPDH − CtPTK2). Each dot in the graph represents the value of one biological sample. The solid red lines show the median and the solid black lines represent the interquartile range. *p ≤ 0.05.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Hybridization

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 8. hAM homogenate inhibits the expression of proteins involved in actin cytoskeleton reorganisation. (A) Western blot analysis of cortactin, RhoA, RhoC, Cdc42, Rac1/2/3 and phosphorylation of cortactin in T24, RT4 and NPU cells treated for 24 h with hAM homogenate, hAM extract or appropriate culture medium (controls) without hAM preparations. (B–D) Quantification of relative protein expression and phosphorylation. The expression and phosphorylation of the target proteins were normalised to the α-tubulin values. In addition, the expression and phosphorylation of the target proteins in the treated samples were normalised to the control samples (culture medium without hAM preparation) and these values were presented as log2 fold change (log2FC). The results are as median (solid red line) with data range (minimum and maximum). The dashed black lines represent the normalised value of the control sample (0). Results were obtained on the basis of 3–6 biological replicates. The most representative blot of the mean effect is presented. The original blots are presented in Supplementary Fig. 2. The absence of full-length membranes is due to the cropping of the membranes prior to hybridization with primary antibodies. *p ≤ 0.05.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Control, Hybridization

Journal: Oncogene

Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

doi: 10.1038/onc.2014.97

Figure Lengend Snippet: (a) Western blot analysis of proteins involved in unfolded protein response and ER stress response. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU2) and two different siRNAs targeting VCP (siVCP1 and siVCP2). After 72 hrs of transfection, cells were harvested and analyzed for protein expression. (b) Western Blot analysis of proteins involved in ER stress response. A549 cells were treated with vehicle or with indicated concentrations of Thapsigargin (TH). (c) Western Blot analysis of proteins involved in ER stress response. A549 cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU2). 48 hrs post siRNA transfections cells were treated with vehicle or with 20nM of Thapsigargin (TH) for 30 min and harvested 24 hrs later. (d) UBQLN1 loss does not alter ERAD. 293T cells stably expressing TCRyfp, a well-known ERAD substrate, were untreated (NT), treated with MG132, or transfected with the indicated siRNA. Cells were subjected to flow cytometry 24 hrs after MG132 treatment or 72 hrs post siRNA transfection to determine the intensity of yfp signal. MG132 treatment is used as a positive control, as ERAD is known to rely on the proteasome for destruction of substrates.

Article Snippet: Human non-small cell lung carcinoma cell line A549 and H358 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Western Blot, Transfection, Expressing, Stable Transfection, Flow Cytometry, Positive Control

Journal: Oncogene

Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

doi: 10.1038/onc.2014.97

Figure Lengend Snippet: (a) Genes differentially expressed upon UBQLN1 knockdown in A549 cells. Comparison of gene expression profiles between A549 cells transfected with either siU1 or siU1-2 and siNT identified a set of 320 unique overlapping genes differentially expressed upon UBQLN1 knockdown (FDR<0.05, fold-change > or < 2). (b) Expression levels of the 320 UBQLN1 regulated genes in siU1, siU2 and siNT treated A549 cells. Genes are arranged in descending order of the average fold change of siU1 and siU1-2 treated cells vs siNT. Expression values are displayed scaled to the maximum expression for each individual gene across the samples. (c) Cellular functions and disease processes enriched within the UBQLN1 gene signature. IPA analysis of the 320 UBQLN1 regulated genes identified cellular movement as the most significantly enriched function. The top 15 diseases and disorders, molecular and cellular functions, and physiological development and function processes are indicated (B-H corrected p-value <0.01).

Article Snippet: Human non-small cell lung carcinoma cell line A549 and H358 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Knockdown, Comparison, Gene Expression, Transfection, Expressing

Journal: Oncogene

Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

doi: 10.1038/onc.2014.97

Figure Lengend Snippet: (a) Migration assay in A549 and H358 cells. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU1-2). 24 hrs post siRNA transfections a pipette tip was used to scratch the dish to make a “wound”. Cells were examined after wound has been formed and successively for 24hr and 48hr post wound formation and photographed. (b) Invasion assay in A549 cells. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU1-2). 24 hours after siRNA transfection cells were seeded into Boyden chambers without (left) or with (right) matrigel. The lower chamber contained media with serum, whereas the upper chamber containing the cells was without serum. 48 hrs later cells on the underside of the membrane were fixed and stained. (c) Quantification of relative number of cells migrated or invaded through matrigel (*P < 0.05).

Article Snippet: Human non-small cell lung carcinoma cell line A549 and H358 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Migration, Transfection, Transferring, Invasion Assay, Membrane, Staining

Journal: Oncogene

Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

doi: 10.1038/onc.2014.97

Figure Lengend Snippet: (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.

Article Snippet: Human non-small cell lung carcinoma cell line A549 and H358 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Transfection, Expressing, Fluorescence, Staining, Knockdown

Journal: Oncogene

Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

doi: 10.1038/onc.2014.97

Figure Lengend Snippet: (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

Article Snippet: Human non-small cell lung carcinoma cell line A549 and H358 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Expressing, Western Blot, Transfection, Knockdown, Fluorescence, Staining

Journal: Oncogene

Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

doi: 10.1038/onc.2014.97

Figure Lengend Snippet: (a) UBQLN1 is frequently lost and underexpressed in cancer cell lines. Cancer cell lines are separated by UBQLN1 copy number status and their corresponding UBQLN1 expression plotted. Box-plots depict the median group expression (middle line), the 25th and 75th percentiles (bottom and top of box, respectively), with the whiskers indicating the 10th and 90th percentiles. Values for all samples outside this range are represented by dots. The UBQLN1 locus is lost in 243 of the 877 cell lines examined. UBQLN1 is significantly under-expressed in samples with UBQLN1 loss or deletion (p<0.0001, Mann-Whitney U-Test). (b) UBQLN1 is frequently lost and underexpressed in human lung adenocarcinomas. Samples are plotted as in A. The UBQLN1 locus is lost in 117 of 230 lung cancers examined. UBQLN1 is significantly underexpressed in tumors with loss or deletion (p<0.0001, Mann-Whitney U-Test). (c) Cancer relevant nonsense mutations in UBQLN1 are degraded in a proteasome dependent manner. Two nonsense mutations found in UBQLN1 in human cancers (UBQLN1 G573X and UBQLN1 G499X ) were introduced into wild-type UBQLN1 (UBQLN1 wt ). Constructs expressing the indicated gene or an empty vector (V) were transfected into 293t cells and 36 hours post transfection they were treated with MG132 for the indicated times. GFP is expressed from the same mRNA as the UBQLN1 genes following and IRES. (d) Recurrent mutations observed in both UBQLN1 and UBQLN2 which have been identified in human lung cancers. (e) Loss of UBQLN2 induces EMT. UBQLN2 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN2 (siU2-5, siU2-6). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. Arrow indicates the UBQLN2 band.

Article Snippet: Human non-small cell lung carcinoma cell line A549 and H358 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Expressing, MANN-WHITNEY, Construct, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Diet High in Soybean Oil Increases Susceptibility to Colitis in Mice

doi: 10.1101/2021.02.08.430328

Figure Lengend Snippet: A) Bacterial species plots of intestinal epithelial cells (IECs) from the small and large intestines (N=4–5) of mice fed Viv chow or a diet enriched in soybean oil, with no added fiber (SO). B) Phenotypic characterization of the E. coli isolate enriched by SO (mAIEC), compared with the human AIEC LF82 and the noninvasive E. coli K12. Assessments were made for bacterial adherence to CaCo-2BBe cells, intracellular invasion of CaCo-2BBe cells, and replication in J774A.1 murine macrophages. * P < 0.05, one-way ANOVA, Tukey’s post hoc comparison. N=12 across 4 experiments. C) Correlation between indicated mouse metadata and log10 of mAIEC relative abundance in the small and large IECs of mice fed Viv or SO+f diets. Pearson correlation coefficient (r) and goodness of fit or R 2 values for linear regression are indicated on the graphs. P value of the correlations ( P) is 0.0007 for body weight, 0.001 for mesenteric fat, 0.006 for subcutaneous fat, 0.005 for retroperitoneal fat, 0.002 for gonadal fat, <0.001 for liver as percent body weight and 0.05 for colon length.

Article Snippet: Control bacteria were the LF82 human AIEC (kindly provided by the late Dr. Arlette Darfeuille-Michaud) and K12 (a noninvasive E. coli , ATCC 25404).

Techniques: Comparison

Journal: Cell Death & Disease

Article Title: USP15 negatively regulates lung cancer progression through the TRAF6-BECN1 signaling axis for autophagy induction

doi: 10.1038/s41419-022-04808-7

Figure Lengend Snippet: A , B USP15KO A549 ( A ) and USP15KO H1299 ( B ) lung cancer cells were generated using CRISPR/Cas9 gene-editing method as described in “Materials and methods”. USP15 KO A549 or USP15 KO H1299 colonies were selected and confirmed by western blots with anti-USP15 or anti-GAPDH antibodies. C , D Control (Ctrl) A549 and USP15 KO A549 cells were suspended in culture medium. Cells were added to the upper compartment of a 24-well Transwell® chamber containing polycarbonate filters with 8-mm pores and coated with 60 mL of Matrigel. Culture medium with 10% FBS was added to the lower chamber. After incubating for 24 h. Cells in the upper compartment were removed, washed with PBS, and fixed. Invaded cells were stained with 4,6-diamidino-2-phenylindole ( C ) and quantified by counting the number of cells ( D ). Results are presented as mean ± SEM of three independent experiments. ** P < 0.01. E , F Ctrl H1299 and USP15 KO H1299 cells were suspended in culture medium. Cells were added to the upper compartment of a 24-well Transwell® chamber. An invasion assay was then performed. Cells were fixed and stained ( E ). The number of migrated cells was counted ( F ). Results are presented as mean ± SEM of three independent experiments. * P < 0.05.

Article Snippet: A549 cells (human lung cancer cell line; ATCC, CCL-185) and H1299 cells (human non-small cell lung cancer cell line; ATCC, CRL-5803) were maintained in RPMI 1640 medium (Sigma-Aldrich, 31800-022) supplemented with 10% FBS, penicillin (100 μg/mL), and streptomycin (100 μg/mL) in a 5% CO 2 humidified atmosphere at 37 °C.

Techniques: Generated, CRISPR, Western Blot, Control, Staining, Invasion Assay

Journal: Cell Death & Disease

Article Title: USP15 negatively regulates lung cancer progression through the TRAF6-BECN1 signaling axis for autophagy induction

doi: 10.1038/s41419-022-04808-7

Figure Lengend Snippet: A , B Control (Ctrl) and USP 15KO H1299 cells were seeded into 12-well cell culture plates. Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip. The wound was then treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), CQ (10 μM) or 3-MA (5 mM) plus LPS (10 μg/mL) for different time periods as indicated. A representative experiment was shown ( A ). The residual gap between migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area (±SEM, n = 3) ( B ). * P < 0.05; *** P < 0.001. C , D Ctrl and USP15 KO H1299 cells were suspended in RPMI medium including vehicle, LPS (10 μg/mL), CQ (10 μM) or 3-MA (5 mM) plus LPS (10 μg/mL) and placed on top chambers of 24-transwell plates. After overnight incubation, cells were fixed and stained with crystal violet ( C ). The number of migrating cells was counted. Results are presented as mean ± SEM of three independent experiments ( D ). * P < 0.05; ** P < 0.01. E – H Ctrl and USP15 KO H1299 cells were treated without or with 10 μg/mL LPS as indicated. Total RNA was extracted, cDNA was synthesized, and RT−qPCR analysis was performed with specific primers for MMP2 ( E ), CCL2 ( F ), CCL20 ( G ), and IL-6 ( H ). Results are presented as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. I A schematic model of the induction of autophagy induced by TLR4, thereby enhancing migration and invasion of USP15 KO lung cancer cells.

Article Snippet: A549 cells (human lung cancer cell line; ATCC, CCL-185) and H1299 cells (human non-small cell lung cancer cell line; ATCC, CRL-5803) were maintained in RPMI 1640 medium (Sigma-Aldrich, 31800-022) supplemented with 10% FBS, penicillin (100 μg/mL), and streptomycin (100 μg/mL) in a 5% CO 2 humidified atmosphere at 37 °C.

Techniques: Control, Cell Culture, Sterility, Transferring, Incubation, Staining, Synthesized, Quantitative RT-PCR, Migration

Journal: Cell Death & Disease

Article Title: USP15 negatively regulates lung cancer progression through the TRAF6-BECN1 signaling axis for autophagy induction

doi: 10.1038/s41419-022-04808-7

Figure Lengend Snippet: A , B A549 ( A ) and H1299 ( B ) lung cancer cells were transiently transfected with FLAG-USP15. Immunoprecipitation (IP) assay was performed with anti-Ig or anti-FLAG antibodies, and then immune-probed with anti-FALG and anti-BECN1 antibodies, as indicated. C Immunoprecipitation (IP) assay was performed with anti-FLAG antibody using HEK 293T cells transfected with mock, FLAG-BECN1, HA-UB, and MYC-USP15 as indicated. Immunoblotting (IB) assay was performed with anti-FLAG or anti-HA antibody. D IP assay was performed with anti-FLAG antibody using HEK 293T cells transfected with mock, FLAG-BECN1, HA-Ub, MYC-USP15 wild-type (WT), MYC-USP15 C269A, and MYC-USP15 H862A as indicated. IB assay was performed with anti-FLAG or anti-HA antibody. E A schematic model of how USP15 induces deubiquitination of BECN1. In the presence of USP15, UPS15 interacts with BECN1 and induces deubiquitination of BECN1, leading to the inhibition of autophagy (indicated as a red arrow). In the absence of USP15, in contrast, TRAF6 interacts with BECN1 and induces ubiquitination of BECN1, leading to the induction of autophagy (indicated as a black arrow).

Article Snippet: A549 cells (human lung cancer cell line; ATCC, CCL-185) and H1299 cells (human non-small cell lung cancer cell line; ATCC, CRL-5803) were maintained in RPMI 1640 medium (Sigma-Aldrich, 31800-022) supplemented with 10% FBS, penicillin (100 μg/mL), and streptomycin (100 μg/mL) in a 5% CO 2 humidified atmosphere at 37 °C.

Techniques: Transfection, Immunoprecipitation, Western Blot, Inhibition, Ubiquitin Proteomics