Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Clinical protocol for CAR T-cell infusion (CTL019). B. Blood T cell counts from patients (n = 15). Red dashed line, the mean ; blue line, total blood lymphocyte counts. C & D. Visualization of early sequestration (0–1 hr) of hHER2 CAR-T cells after adoptive transfer with PET/CT (89Zr-DFO-labeled T cells) (C) or IV-MPM (red, dextran-labeled vessels; green, GFP-expressing CAR-T cells; blue, SHG) (D). IV-MPM images from the lung at the indicated times post-injection (D). Scale bars, 50 μm. E. Representative 3D imaging of CAR-T cell accumulation in cleared lung 4 and 72 hr after i.v. injection (red, blood vessels; green, CAR-T cells). Scale bars, 1 mm. F. The cell density curve and histogram of the distribution of T cells extravasated from the nearest blood vessel (attraction distance, AD). G. 3D image of GFP-expressing CAR-T cells and CD31-labeled vessels. H. The number of injected CAR-T cells in the lung, blood, and tumor at the indicated times. I. in vitro migration of hHER2-CAR T cell (red) to hHER2-B16-BFP tumor spheroid (blue) and . Scale bars, 100 μm. J. CAR-T cells from the lung 72 hr after i.v. injection (T72; CFSE) and newly in vitro activated CAR-T cells (T0; CTFR) were co-transferred (1:1 mix) and the fold change in the homing index was analyzed 24 hr after injection (n = 4, 13 mice per experiment). K. Volcano plot and signaling pathway from RNA-seq analysis in CAR-T cells (T72 vs. T0). L. Percentage of Ki67+ cells in vitro activated CAR-T cells (0 hr) or CAR-T cells isolated from the lung after i.v. injection (4 and 72 hr). M. Flow cytometry results of PD-1 and Tim-3 expression levels on naïve and CAR-T cells isolated from the lung at indicated times post-injection. N. The cytotoxicity of CAR-T cells (PBS, T72, and T0) against B16-hHER2. O. The proportion of CAR-T cells distributed across different organs post-injection. Representative image of 5 mice (D-E). Representative data from three experiments with n = 3 per group (L-N) n = 3 mice per group (F, O). Results are shown mean ± SEM (H, J, L, N). The data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparison posttest (H) or two-tailed Student’s t test (N).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Adoptive Transfer Assay, Positron Emission Tomography-Computed Tomography, Labeling, Expressing, Injection, Imaging, In Vitro, Migration, RNA Sequencing, Isolation, Flow Cytometry, Comparison, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Left: Deep 3D imaging (1 mm thick) of CAR-T cell accumulation in the cleared mouse lung after 72 hr after i.v. injection (red; CD11c (top) or MHCII (bottom), green; CAR-T cell (GFP)). Right: Graph shows colocalization of red signal versus GFP (CAR-T cells). Pearson coefficient was generated as Green (CAR-T cell) /Red (anti-CD11c or anti-MHCII). Data are presented as mean ± SEM; n = 3 mice/group. B. Flow cytometric analysis of OT-I T cells in the lung, blood, and tumor after i.v. injection (4, 24, 48, and 72 hr; mean ± s.e.m., n = 3 mice per group). C. PCA of differentially expressed genes in CAR-T cells (T72 vs. T0). D. Gating strategy for detection of hHER2-CAR-T cells from organs (lungs) E. Expression of Ki67+ in CAR-T cells isolated from the lung after i.v. injection. F. Relative expression of IFN-γ and TNF-α in CAR-T cells isolated from lung at indicated times after i.v. injection. Data are presented as mean ± SEM; n = 3 mice/group. G. Representative flow cytometry results show cell surface PD-1 and Tim-3 expression levels in OT-I naïve T cells, freshly activated in vitro (0 hr), or isolated from the lung after 72 hr post-injection. H. hHER2-CAR-T cells isolated from the lung or dLN after 72 hr i.v. injection were cocultured with B16-hHER2 for 24 hr. Dead B16 cells were stained with 7-amino-actinomycin D (7-AAD). The data reflect 3 independent experiments (the mean ± SEM, n = 3). The data were analyzed by two-tailed Student’s t test (*P = 0.002). I. Representative flow cytometry results showing cell surface PD-1 and Tim-3 expression levels in naïve and CAR-T cells isolated from the lung at the indicated times post-injection.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Imaging, Injection, Generated, Expressing, Isolation, Flow Cytometry, In Vitro, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Schematic for the development of a pooled in vivo CRISPR screening platform in mouse CD8 T cells (Created with BioRender.com) B. Rank-ordered norm z-score of the beta values for all three OT-I T cell distribution screens in B16-OVA bearing mice (yellow, migratory genes; blue, sequestered genes). The top three sequestered and the top five migratory genes are indicated. C. Genes rank based on the difference in beta scores across sequestered and migratory conditions. D. Sectored scatter plot of gene-level beta values from Tsequestered vs. Tmigratory cell populations isolated from B16-OVA bearing mice. Significant migratory genes and sequestered gene signatures (FDR < 5%) are colored yellow and blue, respectively. Ellipse (gray dashed lines), 5% FDR threshold delineated by normal ellipse fit to differential scores. E. qPCR of St3gal1 in mouse and human CD8 T cells (naïve vs. activated) as compared to three averaged housekeeping genes (n = 3–11). F. Expression levels of St3gal1 in activated CD8 T cells (from Days 0 to 5). Loading control: β-actin. G. qPCR of Spc24 in mouse and human CD8 T cells (naïve vs. activated) as compared to three averaged housekeeping genes (n = 3–4). H. Expression levels of Spc24 in activated CD8 T cells (from Days 0 to 5). Loading control: β-actin. I. Expression levels of Spc24 and St3gal1 in Cas-9 hHER2 CAR-T cells with sgSpc24 and sgSt3gal1, respectively. Loading control: β-actin. J. The cytotoxicity of CAR-T cells (PBS, sgSpc24 cells, and sgSt3gal1 cells) against B16-hHER2 (n = 3). K. The distribution of CAR-T cells with (WT; CTFR)/without (KO; CFSE) Spc24 or St3gal1 24 hr after injection (n = 3). The fold change in the homing index, as determined by (KOsample/WTsample)/(KOinput/WTinput). Results are shown mean ± SEM (E, G, J-K). The data reflect three independent experiments (J, K) The data were analyzed by two-tailed Student’s t test (E, G).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: In Vivo, CRISPR, Isolation, Expressing, Control, Injection, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A & B. MAL-II binding to CD8 T cells (A) and St3gal1 KO CAR-T cells (B). C. Immunoprecipitation of MAL-II-binding proteins in CD8 T cells. D. Western blot of the activated CD8 T cell lysates after enzyme treatments. In the graph, each bar represents the calculated molecular weight of protein bands. E. Immunoprecipitation of MAL-II binding CD18 in activated CD8 T cells. F. Recruitment of CAR-T cells into lung tissue (n = 3 mice per group). G. The numbers of WT or LFA-1 (CD11a)-KO CAR-T cells 24 hr after CAR-T cells injection (n = 3). H. In vitro CD8 T cell migration on ICAM-1-coated plates. (n = 3, 13–38 individual cells per mouse). I. In vitro activated CD8 T cell migration on ICAM-1-coated plates ± CXCL12. (n = 3, 10–57 individual cells per mouse). J. LFA-1 FRET assay with CD11a-mYFP and CD18-mCFP. Scale bars, 5 μm. (n = 3, 20–40 individual cells per condition). K. The density of microvilli of CD8 T cells by SEM (n = 6–8). Scale bars, 1 μm. L. The ratio of the TIRFM signal from the LFA-1 accumulation of CD8 T cells at sites of contact with the surface (n=3). M. Changes in the endocytosis of LFA-1 in CD8 T cells after treatment with α2,3-neuraminidase (n = 3–4). N. Time course of surface and intracellular LFA-1 expression. O & P. Changes in the endocytosis (O) and cell surface expression (P) of LFA-1 in CAR-T cells. (n = 3). Q & R. The migration of activated CD8 T cells pretreated with Exo1 (Q) or transfected with Rab13 siRNA (R) (n =3, 20–68 individual cells per mouse). S. The cytotoxicity of CAR-T cells against B16-hHER2 (n = 3). T & U. The percentage of hHER2 CAR-T cells in peripheral tissue sites (T) and tumors (U) 24 hours after adoptive transfer (n=3 mice per group). Results are shown mean fluorescence intensities (MFI) ± SEM (A-B) and mean ± SEM (C, F-M, O-P). All data and representative images reflect three independent experiments. Statistical analyses were performed using one-way ANOVA with Bonferroni posttest (A-C, H-I, Q-R), two-sided Mann-Whitney test (F, T-U), two-sided, unpaired Student’s t test (G, J) or two-tailed Student’s t test (K-M, O-P). NS; not significant.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Molecular Weight, Injection, In Vitro, Migration, Expressing, Transfection, Adoptive Transfer Assay, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. LFA-1 immunoprecipitates obtained from human T cells bound on ICAM-1 (IC-1)- or poly-L lysine (PLL)-coated cover glasses. LFA-1-associated proteins were identified by silver staining and mass spectrometry. B. Expression levels of αII-spectrin, βII-spectrin, and MyH9 in activated T cells (from Days 0 to 5). Loading control: β-actin. C. A confocal FRAP experiment with activated CD11a-mYFP CD8+ T cells (n = 3). Scale bars, 1 μm. D. & E. Representative TIRFM images of activated CD11a-mYFP CD8+ T cell adhesion on ICAM-1-coated plates (D) and the area and spreading speed calculated from zone including all TIRF signals for each cell (E). Scale bar, 20 μm. Each dot represents one cell (n = 3). Bar = mean. F. Naïve and activated CD8 T cell migration on ICAM-1- or ICAM-1+CXCL12-coated surfaces. (n = 3. 21–42 individual cells per mouse). G. Schematic illustrating the regulation of LFA-1 endocytic recycling via βII-spectrin or St3gal1 expression (Created with BioRender.com). H. βII-spectrin (GFP+) expression in T cells isolated from GFP-ROSAβII-spectrin mice (TβII-spectrin) after in vitro treatment with TAT-Cre recombinase. I. Cross sections image in the middle of hHER2-CAR T cell (WT and TβII-spectrin)-endothelial cell (bEND.3 cells) contacts by transmission EM. Black arrows, LFA-1 stainings at the cell-cell contacts. Scale bars, 2 μm and 0.1 μm. J. The ratio of the TIRFM signal from the LFA-1 accumulation of hHER2-CAR T cells (WT and TβII-spectrin) at sites of contact with the ICAM-1-coated surface (n = 3). K. Flow cytometry analysis of MAL-II binding to naïve and activated CD8 T cells (WT, TβII-spectrin, and TβII-spectrin + St3gal1-shRNA-treated cells) (n = 3). Representative images or data were collected from three independent experiments (A-B, F, H) or two independent experiments (I). Results are shown the mean ± SEM (C, F) and mean fluorescence intensities (MFI) ± SEM (K). Statistical analyses were performed by ordinary two-way ANOVA (C), two-sided, unpaired Student’s t test (E-F, J, K).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Silver Staining, Mass Spectrometry, Expressing, Control, Migration, Isolation, In Vitro, Transmission Assay, Flow Cytometry, Binding Assay, shRNA, Fluorescence

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Schematic showing the assessment of tissue homing of TβII-spectrin (green) and Tcontrol (red) cells transfected with hHER2-CAR in hHER2 Tg mice bearing B16-hHER2 tumors. B. TβII-spectrin (green) and Tcontrol (red) hHER2-CAR were cotransferred (1:1 mix) and the fold change in the homing index was analyzed at each time point (n = 5 mice per condition). C. Represented deep 3D imaging (1-mm thick) of CAR-T cell accumulation in a cleared mouse lung 72 hr after i.v. injection (blue; blood vessels [CD31], green; TβII-spectrin, red; Tcontrol). Scale bars, 100 μm. D. Tumor growth and final tumor size from hHER2 Tg mice bearing B16-hHER2 tumors infused with TβII-spectrin and Tcontrol cells transfected with hHER2-CAR (n = 10 mice per group). E. Mouse survival curves up to 50 d after tumor inoculations. F. Frequency of CAR-T cells over total lymphocytes in B16-hHER2 tumours, lung, or dLN as measured by flow cytometry on day 22. respectively (n = 4). G. βII-spectrin western blot analysis of CAR-T cell infusion products obtained from 23 BCL patients who showed a complete recovery (CR), partial recovery (PR), or progressive disease (PD). Representative male and female samples from each patient group are shown. H. Quantification of total βII-spectrin protein expression in CAR-T cell products from patients with CR, PR, or PD as determined by western blotting. I. Correlation between βII-spectrin expression in CAR-T cell products and patient neurotoxic effects and cytokine release syndrome. Pearson correlation calculations were performed for linear correlations. J. Luminex assay of the proinflammatory cytokines IFN-γ, IL-6 and IL-1β in patient serum before (Day −5) and after (Day 2) CAR-T cell infusion. The data represent the mean ± SEM (B, D, F, H). Statistical analyses were performed by ordinary two-way ANOVA (D), two-sided, unpaired Student’s t test (B, D, H) or two-sided log-rank Mantel–Cox test (E). Whiskers represent maximum and minimum values (D, F, H).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Transfection, Imaging, Injection, Flow Cytometry, Western Blot, Expressing, Luminex

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

doi: 10.1186/s13046-020-01808-3

Figure Lengend Snippet: HCC-TCM promotes M2 polarization via Wnt2b/β-catenin signalling. a THP-1 derived macrophages (THP-1-M) were incubated with 50% HCC-TCM for 48 h to obtain the HCC-educated macrophages (HCC-TAMs). Transcript expression levels of Wnt2b were determined in HCC-TAMs by qPCR. b The expression levels of Wnt2b (red) in CD68 + macrophages (green) were determined by immunofluorescence using TMA containing pairs of tumors and matched para-carcinoma tissues of HCC patients. c , d THP-1-M were transfected with control vectors or Wnt2B-V5 (over-Wnt2b) vectors for 48 h. The expression levels of CD163 and markers for M1 or M2 macrophages on/in these cells were determined by flow cytometry and qPCR, respectively. THP-1-M infected with control vectors, sh-Wnt2b or sh-CTNNB1 (β-catenin) vector were acquired as described in the Materials and Methods, and then incubated with 50% HCC-TCM for 48 h. The expression levels of CD163 and markers for M1 or M2 macrophages on/in these cells were determined by flow cytometry ( e , i ) and qPCR ( f , j ), respectively. The expression levels of β-catenin in HCC-TAMs that were infected with control vectors or sh-Wnt2b vectors were determined by western blotting and immunofluorescence respectively ( g , h ). One representative of at least three independent experiments is shown. qPCR, quantitative real-time PCR; HCC, hepatocellular carcinoma; TCM, tumour condition culture medium; TAMs, tumour-associated macrophages; 7721, SMMC-7721; TMA, Tissue microarray. Data are presented as mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: The human beta-catenin (CTNNB1) shRNA lentivirus vectors pLKO.1-sh-β-catenin and its control vectors were purchased from Addgene (Plasmid #19761).

Techniques: Derivative Assay, Incubation, Expressing, Immunofluorescence, Transfection, Control, Flow Cytometry, Infection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

doi: 10.1186/s13046-020-01808-3

Figure Lengend Snippet: The activation of Wnt2b/β-catenin signalling enhances TAMs-induced tumour-promoting effects. a THP-1 derived macrophages (THP-1-M) were transfected with control vectors or Wnt2B-V5 (over-Wnt2b) vectors for 48 h. These macrophages were incubated with RPMI 1640 for an additional 24 h to obtain the condition medium (CM). HCC cells were cultured in the presence of indicated CM for 48 h. The expression levels of EMT markers were determined by western blotting. ( b-e ) THP-1-M infected with control vectors, sh-Wnt2b or sh-CTNNB1 (β-catenin) vectors were acquired as described in Materials and Methods. These macrophages were incubated with 50% HCC-TCM for 48 h for the preparation of the different TAMs. These TAMs were incubated with RPMI 1640 for another 24 h to obtain the CM. b , c HCC cells were cultured in the presence of the indicated CM for 48 h. The expression levels of EMT markers were determined by western blotting. d HCC cells were incubated with culture medium (Ctrl) or the indicated CM for 24 h. The cell viability of each group was detected by MTT assay. e HCC cells were scratched with a plastic pipette tip and incubated with culture medium (Ctrl) or indicated CM for 24 h. The results of this wound healing assay were photographed and measured. One representative of at least three independent experiments is shown. qPCR, quantitative real-time PCR; HCC, hepatocellular carcinoma; TCM, tumour condition culture medium; TAMs, tumour-associated macrophages; 7721, SMMC-7721. Data are presented as mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: The human beta-catenin (CTNNB1) shRNA lentivirus vectors pLKO.1-sh-β-catenin and its control vectors were purchased from Addgene (Plasmid #19761).

Techniques: Activation Assay, Derivative Assay, Transfection, Control, Incubation, Cell Culture, Expressing, Western Blot, Infection, MTT Assay, Transferring, Wound Healing Assay, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: PGC-1α overexpression enhances expression of LARS1 in HEK293 cells. ( A ) Total RNAs isolated from PGC-1α- or pcDNA-expressing HEK293 cells were used for ACP-based GeneFishing analysis as described in Materials and Methods. Amplified bands showing different densities between two cell lines were re-amplified and sequenced for gene annotation. Arrow indicates DEG band identified as LARS1. ( B ) Left: The mRNA levels of PGC-1α and LARS1 were examined by qRT-PCR. β-actin is used as an endogenous control. Middle and Right: The protein levels of PGC-1α and LARS1 in control pcDNA-HEK293 and PGC-1α-HEK293 cells. Protein lysates were prepared and subjected to Western blot analysis as described in Materials and Methods. Equal protein loading was ensured by showing uniform β-actin expression. Densitometry results are indicated above the bands. Representative data of three independent experiments are shown. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, pcDNA-HEK293 cells. ( C , D ) Left panel: Immunofluorescence staining was performed as described in Materials and Methods using anti-PGC-1α (( C ), green) and anti-LARS1 (( D ), green) antibodies. Right panel: Mean Fluorescence Intensity (MFI) of PGC-1α ( C ) and LARS1 ( D ) of immunofluorescence images were quantified using Image J software (v.1.53t). GP, arbitrary general primer; 1, control HEK293 cells; 2, PGC-1α-HEK293 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images .

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Over Expression, Expressing, Isolation, Amplification, Quantitative RT-PCR, Control, Western Blot, Immunofluorescence, Staining, Fluorescence, Software

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: LARS1 overexpression enhances cell proliferation, migration, and invasion of SW480 cells. ( A ) Left panel: Expression levels of PGC-1α and LARS1 mRNA in pcDNA-, PGC-1α-1-, or -2-SW480 cells. Middle panel: Expression of PGC-1α and LARS1 in SW620 and SW480 cells. Right panel: SW480 cells were transfected with LARS1 or pCMV6 expression vector and screened based on their resistance to G418 (800 μg/mL). Western blot was used to detect PGC-1α and LARS1. β-actin was used as an internal control. Densitometry results are indicated above bands. Representative data of three independent experiments are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pcDNA-SW480 cells. ( B , C ) pCMV6-, LARS1-2, or -4-SW480 cells were seeded and cultured for the indicated times and cell proliferation was determined by cell counting ( B ) and MTT assay ( C ). Data are presented as the mean ± SD of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. ( D , E ) Left panel: Representative figures of pCMV6-, LARS1-2-, and -4-SW480 cells in the transwell migration assay ( D ) and transwell invasion assay ( E ) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells ( D ) and transmembrane invaded cells ( E ) were counted for five randomly chosen visual fields. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pCMV6-SW480 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images .

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Over Expression, Migration, Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Cell Culture, Cell Counting, MTT Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: LARS1 knockdown reduces cell proliferation, migration, and invasion of SW620 cells. ( A ) Left panel: Expression levels of PGC-1α and LARS1 mRNA in NC shRNA-, PGC-1α shRNA-1-, or -2-SW620 cells. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001, NC shRNA-SW620 cells. Middle panel: SW620 cells were transfected with NC shRNA or LARS1 shRNA expression vector and screened based on their resistance to G418 (800 μg/mL). Western blot was used to detect PGC-1α and LARS1. β-actin was used as an internal control. Densitometry results are indicated above bands. Representative data of three independent experiments are shown. Right panel: Data are expressed as the mean ± SD of three independent experiments. * p < 0.05 vs. NC shRNA-SW620 cells. NS, not significant. ( B , C ) NC shRNA-, LARS1 shRNA-4-, or -5-SW620 cells were seeded and cultured for the indicated time and cell proliferation was determined by cell counting ( B ) and MTT assay ( C ). Data are presented as the mean ± SD of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. ( D , E ) Left panel: Representative figures of NC shRNA- and LARS1 shRNA-5-SW620 cells in the transwell migration assay ( D ) and transwell invasion assay ( E ) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells ( D ) and transmembrane invaded cells ( E ) are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images .

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Knockdown, Migration, Expressing, shRNA, Transfection, Plasmid Preparation, Western Blot, Control, Cell Culture, Cell Counting, MTT Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: PGC-1α regulates cell proliferation, migration, and invasion of SW480 and SW620 cells by regulating LARS1 expression. ( A – C ) PGC-1α-1-SW480 cells were transiently transfected with NC shRNA vector or LARS1 shRNA expression vector, respectively. ( A ) After transfection, the cell counts of each type of cell are presented as the mean ± SD of three separate experiments. ( B , C ) Left panel: Representative figures of pcDNA-, PGC-1α-1/NC shRNA-, and PGC-1α-1/LARS1 shRNA-SW480 cells in the transwell migration assay ( B ) and transwell invasion assay ( C ) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells ( B ) and transmembrane invaded cells ( C ) are shown. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 vs. pcDNA-SW480 cells. ## p < 0.01, ### p < 0.001 vs. PGC-1α-1/NC shRNA-SW480 cells. ( D – F ) PGC-1α shRNA-1-SW620 cells were transiently transfected with pCMV6 expression vector or LARS1 expression vector, respectively. ( D ) After transfection, the cell counts of each type of cell are presented as the mean ± SD of three separate experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. PGC-1α shRNA-1/pCMV6-SW620 cells. ( E , F ) Left panel: Representative figures of NC shRNA-, PGC-1α shRNA-1/pCMV6-, and PGC-1α shRNA-1/LARS1-SW620 cells in the transwell migration assay ( E ) and transwell invasion assay ( F ) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells ( E ) and transmembrane invaded cells ( F ) are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. PGC-1α shRNA-1/pCMV6-SW620 cells. ( G – I ) PGC-1α shRNA-1-SW620 cells were transiently transfected with NC shRNA expression vector or LARS1 shRNA expression vector, respectively. ( G ) After transfection, the cell counts of each type of cell are presented as the mean ± SD of three separate experiments. ( H , I ) Left panel: Representative figures of NC shRNA-, PGC-1α shRNA-1/NC shRNA-, and PGC-1α shRNA-1/LARS1 shRNA-SW620 cells in the transwell migration assay ( H ) and the transwell invasion assays ( I ) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells ( H ) and transwell-membrane invaded cells ( I ) are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. NS, not significant.

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Migration, Expressing, Transfection, shRNA, Plasmid Preparation, Transwell Migration Assay, Transwell Invasion Assay, Membrane

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: Expression levels of PGC-1α, LARS1, and several signaling molecules in pcDNA-, PGC-1α-, pCMV6-, LARS1-SW480, NC shRNA-, PGC-1α shRNA-, LARS1 shRNA-SW620 cells. ( A ) (Left panel) Representative three independent Western blot analysis results of pcDNA-, PGC-1α-1-, PGC-1α-2-SW480 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pcDNA-SW480 cells. ( B ) (Left panel) Representative three independent Western blot analysis results of pCMV6-, LARS1-2-, LARS1-4-SW480 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above the bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. ( C ) (Left panel) Representative three independent Western blot analysis results of NC shRNA-, PGC-1α shRNA-1-, PGC-1α shRNA-2-SW620 cells are shown. β -actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. ( D ) (Left panel) Representative three independent Western blot analysis results of NC shRNA-, LARS1 shRNA-4-, LARS1 shRNA-5-SW620 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images .

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Expressing, shRNA, Western Blot, Control

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: LARS1 enhances cell proliferation, migration, and invasion through AKT activation in SW480 cells. ( A , B ) LARS1-2-SW480 cells were treated with/without 0.5 μM AKT inhibitor IV for indicated time ( A ) or for 48 h ( B ) and cell proliferation was measured by cell counting ( A ) and MTT assay ( B ), respectively. ( C , D ) LARS1-2-SW480 cells were treated with/without 0.5 μM AKT inhibitor IV for 48 h. Transwell migration assays ( C ) and invasion assays ( D ) were performed as described in Materials and Methods. ( C ) Left panel: Representative figures of pCMV6-, LARS1-2-SW480 cells treated without/with 0.5 μM AKT inhibitor IV in the transwell migration assay are shown (×200 magnification). Right panel: The number of transmembrane migrated cells was counted for five randomly chosen visual fields. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pCMV-6-SW480 cells; ### p < 0.001 vs. LARS1-2-SW480 cells. ( D ) Left panel: Representative figures of pCMV6-, LARS1-2-SW480 cells treated without/with 0.5 μM AKT inhibitor IV in the transwell invasion assay are shown (×200 magnification). Right panel: The number of transmembrane migrated cells was counted for five randomly chosen visual fields. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pCMV-6-SW480 cells; ### p < 0.001 vs. LARS1-2-SW480 cells. ( E ) After treatment with AKT inhibitor IV, protein lysates were prepared and used for Western blot analysis with corresponding antibodies. β-actin was used as a loading control. (Left panel) The blot is representative of three separate experiments. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LARS1-2-SW480 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images .

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Migration, Activation Assay, Cell Counting, MTT Assay, Transwell Migration Assay, Transwell Invasion Assay, Western Blot, Control

Journal: Cancers

Article Title: PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

doi: 10.3390/cancers15010159

Figure Lengend Snippet: Potential molecular mechanism by which PGC-1α regulates cell proliferation, migration, and invasion of human colorectal cancer cells. In summary, PGC-1α regulates cell proliferation, migration, and invasion via regulation of LARS1/AKT/GSK-3β/β-catenin axis.

Article Snippet: To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures.

Techniques: Migration

Journal: Journal of Biological Chemistry

Article Title: SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

doi: 10.1074/jbc.m802187200

Figure Lengend Snippet: FIGURE 3. Lentivirus-mediated knockdown of SIRT1 diminishes the basal and polyphenol-induced AMPK activation in HepG2 cells. HepG2 cells wereinfectedwithoutorwithlentivirusexpressingeitheranshRNAcontrolor SIRT1 shRNA and then selected in 0.6 g/ml puromycin. Cells were allowed to recover from the selection for 1 week prior to the experiments. A, co-expres- sion of GFP in both control shRNA cells and SIRT1 shRNA cells was observed under fluorescence microscopy. B, endogenous SIRT1 expression was largely suppressed by lentivirus expressing SIRT1 shRNA. Representative immunob- lots for the expression of SIRT1 and -actin are shown in duplicates under the identical condition. C–E, knockdown of SIRT1 by lentivirus-mediated SIRT1 shRNA down-regulates the basal and polyphenol-stimulated AMPK and ACC phosphorylation. HepG2 cells expressing either control or SIRT1 shRNA were quiesced in serum-free medium overnight and treated with S17834 (10 M, 1 h). *, p 0.05 versus untreatment in cells expressing control shRNA; #, p 0.05 versus S17834 treatment in cells expressing control shRNA (mean S.E., n 3).

Article Snippet: The produced expression cassette was inserted into a directional pENTR/D-topo vector (Invitrogen) and then transferred to shRNA expressing lentivirus vector pDSL_hpUGIP (ATCC) by LR-clonase (Invitrogen).

Techniques: Knockdown, Activation Assay, shRNA, Selection, Control, Fluorescence, Microscopy, Expressing, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

doi: 10.1074/jbc.m802187200

Figure Lengend Snippet: FIGURE 8. Proposed scheme for the role of SIRT1-activating polyphenols in the regulation of AMPK signaling and hepatocyte lipid metabolism. SIRT1-activating polyphenols, such as resveratrol and S17834, stimulate LKB1 phosphorylation as well as AMPK phosphorylation and activation. Similarly, overexpression of wild type SIRT1 also increases AMPK phosphorylation, which in turn increases ACC phosphorylation and inhibits ACC activity. As a consequence, decreased production of malonyl-CoA results in up-regulation of fatty acid oxidation as well as down-regulation of fatty acid synthesis, thereby leading to hepatocyte lipid reduction. On the other hand, activation of AMPK by polyphenols inhibits glucose-induced expression of FAS, which contributes to the reduction in triglycerides through inhibition of fatty acid biosynthesis. Moreover, SIRT1 is required for the effects of polyphenols on AMPK and lipids, since the beneficial effects of polyphenols are mimicked by overexpression of wild type SIRT1 and abrogated by inhibition of SIRT1, such as the inactive SIRT1 mutant (SIRT1H355A), shRNA SIRT1, or inhibitors of SIRT1. Importantly, the stimulation of AMPK and the lipid-lowering effect of SIRT1 are abolished by DN-AMPK, suggesting that AMPK acts as a novel func- tional downstream regulator for the metabolic effect of SIRT1. In addition, polyphenols and SIRT1 stimulate AMPK via an LKB1-dependent mechanism. Therefore, SIRT1/LKB1/AMPK signaling may be considered a novel molecular mechanism for potential therapeutic effects of polyphenols on hepatic lipid accumulation in diabetes and age-related metabolic disorders.

Article Snippet: The produced expression cassette was inserted into a directional pENTR/D-topo vector (Invitrogen) and then transferred to shRNA expressing lentivirus vector pDSL_hpUGIP (ATCC) by LR-clonase (Invitrogen).

Techniques: Phospho-proteomics, Activation Assay, Over Expression, Activity Assay, Expressing, Inhibition, Mutagenesis, shRNA