Journal: International Journal of Molecular Sciences

Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes

doi: 10.3390/ijms19020479

Figure Lengend Snippet: Trio Contributes to Basal Rac1 Activity and Cell Size ( a ) The CRISPR/Cas9 system was used to create Trio KO human podocytes. Monoclonal populations were selected and tested for Trio expression using immunoblotting. Although the KO was not complete, it decreased Trio expression by 60% (Trio KO #13) or 58% (Trio KO #14) when normalized to calnexin and then to control Cas9. ECL was used for visualization; ( b , c ) Cdc42/Rac interactive binding (CRIB) pulldown (PD) assays revealed that both Trio KO #13 (0.39 ± 0.10, n = 3, ** p < 0.01 compared to control Cas9) and Trio KO #14 (0.24 ± 0.06, n = 3, *** p < 0.001 vs. control Cas9) had decreased Rac1 activity compared to control Cas9. ECL was used for visualization and quantification was done using densitometric analysis. Results were normalized to tubulin and then to control Cas9 cells; ( d ) MTT assay showed that the proliferation rate of Trio KOs did not differ from that of control Cas9 cells between 24 h and 48 h after plating ( y -axis represents the value at 48 h over the value at 24 h). Trio KOs #9 and #19 also had decreased Trio expression (see ) and were used for the MTT assay; ( e , f ) Control Cas9 and Trio KOs were fixed and stained for phalloidin. Trio KOs (2349 ± 114 μm 2 , n = 89 cells from two experiments, *** p < 0.001 vs. control Cas9) were significantly smaller than control Cas9 (3167 ± 140 μm 2 , n = 81 from two experiments). Scale bar in Cas9 photo ( e ) is 10 μm and is the same for Trio KO photo ( g , h ) GFP-alone or GFP-Trio-GEFD1 were expressed in HP and cells were stained for phalloidin. GFP-Trio-GEFD1 expressing cells were larger (3223 ± 131 μm 2 , n = 65 from two experiments, *** p < 0.001 vs. GFP-alone) than GFP-alone expressing cells (2327 ± 142 μm 2 , n = 61 from two experiments). Scale bar in ( g ) is 10 μm.

Article Snippet: The lentiviral Cas9 and sgRNA Trio were from Genecopoeia (Thorold, ON, Canada).

Techniques: Activity Assay, CRISPR, Expressing, Western Blot, Control, Binding Assay, MTT Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes

doi: 10.3390/ijms19020479

Figure Lengend Snippet: Trio affects motility, attachment, and vinculin distribution. ( a ) Control Cas9 and Trio KOs were allowed to grow to confluency, serum starved overnight, and then a scratch wound was created. Images were taken at time 0 and 5 h after; ( b ) Cell motility was calculated from percent wound closure from 0 to 5 h. Control Cas9 cells had a greater wound closure than Trio KOs (control Cas9: 21.4 ± 0.55, Trio KOs: 17.3 ± 0.99, n = 4, * p < 0.05 vs. control Cas9). The yellow lines have been drawn to easily visualize the two sides of the wound. Scale bar in ( a ) is 100 μm; ( c ) The same number of control Cas9 and Trio KOs were plated on laminin 521 and allowed to attach for 1.25 h. Attached cells were quantified by crystal violet as described in Methods. Control Cas9 cells had more attachment than Trio KOs (absorbance values at 550 nm: control Cas9: 0.17 ± 0.01, Trio KO: 0.13 ± 0.01, n = 3–4, * p < 0.05 vs. control Cas9); ( d ) Control Cas9 and Trio KOs were plated overnight on laminin 521, fixed, and stained for phalloidin (red) and vinculin (green). Co-localization is shown in yellow in merged photo. Scale bar in ( d ) is 10 μm. The amount of vinculin at the periphery was quantified; ( e ) Quantification of peripheral vs. central vinculin staining from ( e ) was performed as described in Methods. Trio KOs had more peripheral vinculin than control Cas9 (control Cas9: 0.55 ± 0.02, n = 21 cells counted from two experiments; Trio KO: 0.72 ± 0.04, n = 19 cells counted from two experiments, *** p < 0.001 vs. control Cas9).

Article Snippet: The lentiviral Cas9 and sgRNA Trio were from Genecopoeia (Thorold, ON, Canada).

Techniques: Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes

doi: 10.3390/ijms19020479

Figure Lengend Snippet: TGFβ1 decreases Rac1 activity in Trio KOs, while increasing CdGAP activation in control cells. ( a ) Control Cas9 and Trio KOs were incubated with TGFβ1 (10 ng/mL) for 60 min. Cell lysates were subjected to CRIB PD and immunoblotting for Rac1 and tubulin. ECL was used for visualization; ( b ) Densitometric analysis. Results were first normalized to tubulin and then fold increase of +TGFβ1 over −TGFβ1 was calculated. In control Cas9 cells, TGFβ1 increased Rac1 activity 1.72-fold ± 0.20, n = 4; whereas in Trio KOs, TGFβ1 decreased Rac1 activity 0.59-fold ± 0.13, n = 4. ** p < 0.01 vs. control Cas9; ( c ) HP were treated with TGFβ1 for the indicated times and cell lysates were pulled down with GST-CA-Rac1. Precipitates and lysates were immunoblotted for CdGAP and tubulin and visualized using LiCor; ( d ) Densitometric analysis. Levels of active CdGAP were normalized to CdGAP levels in the total lysate (i.e., input) and then to 0 min. CdGAP activity was increased at 30 min (1.55-fold ± 0.16, * p < 0.05 vs. unstimulated), 45 min (1.56-fold ± 0.21, * p < 0.01 vs. unstimulated), and 60 min (1.53-fold ± 0.12, ** p < 0.05 vs. unstimulated), n = 3–4; ( e ) Control Cas9 cells and Trio KOs were stimulated with TGFβ1 (10 ng/mL, 60 min), cell lysates were immunoblotted for p-p38 and total p38, and protein was visualized using ECL; ( f ) Densitometric analysis. Basal (unstimulated) levels of p-p38 were normalized to tubulin and then to control Cas9 levels. Trio KOs had 0.55 ± 0.28 fold decrease, n = 4, * p < 0.05 versus control Cas9; ( g ) Results were normalized to tubulin and then fold increase of +TGFβ1 over −TGFβ1 was calculated. TGFβ1 increased p-p38 more in Trio KOs than in control Cas9 (control Cas9: 1.54 ± 0.45 fold, Trio KO: 2.71 ± 0.70 fold, n = 4, * p < 0.05 vs. control Cas9).

Article Snippet: The lentiviral Cas9 and sgRNA Trio were from Genecopoeia (Thorold, ON, Canada).

Techniques: Activity Assay, Activation Assay, Control, Incubation, Western Blot

Journal: bioRxiv

Article Title: Cellular transformation by combined lineage conversion and oncogene expression

doi: 10.1101/525600

Figure Lengend Snippet: ( A ) Phase contrast microscope images showing the phenotype and morphology of the cells in the course of conversion of fibroblasts to iHeps at different times points after transduction with a cocktail of three TFs HNF1A, HNF4A and FOXA3 ( Huang et al , 2014 ). ( B ) Generation of highly proliferative iHep cells by transducing iHeps with two pools of liver cancer-specific oncogenic drivers, a list of xenograft experiments in nude mice that were used to test the tumorigenicity of different conditions, and mutation rates of the oncogenic drivers as reported in the COSMIC database for HCC and MYC amplification as reported in . CMT pool contains three oncogenes CTNNB1 T41A , MYC, and TERT, and CMT+sg TP53 pool contains the same oncogenes along with constructs for TP53 inactivation by CRISPR-Cas9. Phase contrast microscope images showing the phenotype and morphology of the cells. Oncogenes are co-transduced with fluorescent reporter mCherry for detection of transduced cells. Oncogene transduction to fibroblasts fails to transform the cells, passaging of oncogene-expressing fibroblasts results in cellular senescence as demonstrated by beta-galactosidase staining and loss of mCherry-positive oncogene-expressing cells from the fibroblast population. Passaging of iHeps without oncogenes results in apoptosis after few passages. Scale bar 1000 μm unless otherwise specified.

Article Snippet: Expression construct for mCherry (#36084), lentiviral Cas9 expression construct LentiCas9-Blast (#52962), a cloning backbone lentiGuide-Puro (#52963), and the constructs for neuronal conversion (Tet-O-FUW-Ascl1, #27150; Tet-O-FUW-Brn2, #27151; Tet-O-FUW-Myt1l, #27152; Tet-O-FUW-NeuroD1, # 30129; pTetO-Ngn2-Puro, #52047; Tet-O-FUW-EGFP, # 30130; FUW-M2rtTA, #20342) were obtained from Addgene.

Techniques: Microscopy, Transduction, Mutagenesis, Amplification, Construct, CRISPR, Passaging, Expressing, Staining

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Ter119 negative cells and Ter119 positive erythroid cells were purified from wild-type mouse bone marrow cells. G4 levels were tested by flow cytometry using the BG4 antibody that specifically recognizes G4. Quantification is on the right. (B) Bone marrow lineage-negative cells were cultured in Epo medium for 2 days. G4 levels were tested on different days using flow cytometry by the BG4 antibody. Quantification is on the right. (C) CD34+ human HSPCs were cultured in Epo medium for 21 days. The levels of G4 were measured by flow cytometry as in B at the indicated time. Cells at day 7, 14, and 21 represent proerythroblasts, polychromatic to orthochromatic erythroblasts, and orthochromatic to mature red blood cells, respectively. (D) Flow cytometric assays of G4 levels in the indicated bone marrow lineage cells purified from wild-type mice. (E) Quantification of D. (F) Gating strategy of various erythroblasts. Populations I to VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, late orthochromatic to reticulocytes, and mature red blood cells, respectively. (G-H) Flow cytometric assay of G4 level in bone marrow erythroid populations I (G) and V (H) from the indicated mice. Quantification is on the right. (I) Bone marrow lineage negative cells from the indicated mice were cultured in Epo medium for 2 days. G4 levels on different days were measured by flow cytometry using BG4 antibody. Quantification is below the histogram. (J) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (K) Quantitative analyses of G4 levels in cells from J using flow cytometric assays. (L) Quantitative analyses of cell death in cells from J using flow cytometric assays. The dead cells are defined as propidium iodide and annexin V double positive. (M) Quantitative analyses of G4 levels in bone marrow mononuclear cells from the patient with DDX41 mutated MDS. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Purification, Flow Cytometry, Cell Culture, Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Epo medium-cultured mouse bone marrow lineage negative HSPCs were treated with 1 μM PDS for the indicated time. Immunofluorescence assays of γ-H2AX were performed, and representative images of the erythroid cells were presented. Scale bar: 5 μm. (B) Flow cytometry assay of the cells in A. (C) Statistical quantification of γH2AX signals in B. (D) Epo medium-cultured mouse bone marrow lineage negative HSPCs were cultured for 1 day, followed by the treatment of 1 μM PDS for 6 hours. Quantitative RT-PCR analyses of indicated ribosome RNAs were performed using different primer sets. (E) Western blotting assays of indicated in cells from D. Actin was used as a loading control. (F) Same as D except that bone marrow lineage negative HSPCs from HBBCre:Ddx41 fl/fl mouse were cultured for 1 day before the quantitative RT-PCR assays. (G) Western blotting assays of the indicated proteins in F. Cells from both day 1 and day 2 cultured cells were analyzed. (H) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (I) Immunohistochemical stains of p53 in bone marrow core biopsies from the patient in normal individual. Scale bar: 100 μm. (J) Quantification of γ-H2AX in bone marrow mononuclear cells from the patient in I and 2 control individuals. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control, Transduction, Expressing, Immunohistochemical staining, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Representative wide-field picture and H&E stains of bone marrow organoid in culture. (B) Whole-mount 3D imaging of the organoids. Imaris was used for cell surface rendering. Organoids were stained with indicated antibodies and subsequently imaged using a laser scanning confocal platform. (C) Confocal immunofluorescence assays of erythroid islands in the iPSC-derived bone marrow organoids (left) and a primary human bone marrow biopsy (right). CD71 was labeled with green for organoids and magenta for primary bone marrow. DAPI: blue. (D) Flow cytometry assays of the organoids using indicated antibodies for various lineages. (E) 10,000 CellVue-labeled donor CD34+ HSPCs were co-incubated with iPSC-derived bone marrow organoids for 3 days in each well of a 96-well plate, followed by an immunofluorescence assay. Representative pictures show the engraftment of donor hematopoietic cells into the organoid. Green, red, and blue represent CD71, CellVue, and DAPI-positive nuclei, respectively. The arrow points to an engrafted CellVue positive cell expressing CD71. (F) Flow cytometry of the organoids using indicated antibodies for various lineages of the engrafted cells in organoids from E. (G) Same as E, except the donor CD34+ cells were transduced with lentiviral vectors expressing Cas9 and indicated sgRNAs before co-incubation. After 3 days, the cells were collected for flow cytometric assays of erythroid and myeloid differentiation of CellVue-positive donor hematopoietic cells and negative iPSC-derived hematopoietic cells. Each data point represents cells combined from 10 organoids. The comparison was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01. (H) Schematic model of the function of DDX41 during erythropoiesis. The diagram is generated through BioRender.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Imaging, Staining, Immunofluorescence, Derivative Assay, Labeling, Flow Cytometry, Incubation, Expressing, Transduction, Comparison, Generated

Journal: EMBO Molecular Medicine

Article Title: Follistatin is a novel therapeutic target and biomarker in FLT 3/ ITD acute myeloid leukemia

doi: 10.15252/emmm.201910895

Figure Lengend Snippet: A In silico analysis (DECipherment of DNA Elements, SABiosciences) and schematic model of transcription factor binding sites on human FST promoter. CBP: CREB‐binding protein; CRE: cAMP‐response element; TSS: transcription start site. B, C The direct binding of p‐CREB to human FST promoter was detected by ChIP‐PCR (B) and ChIP‐qPCR (C). c‐Fos was used as positive control of p‐CREB target gene. Normal IgG was used as negative control of ChIP. D Dual‐luciferase assay demonstrating the direct binding of p‐CREB on human FST promoter. pRL‐CMV, Renilla luciferase vector; pGL‐CRE− and pGL‐CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE−) or wild type (CRE+); p‐GFPSpark, GFP‐expressing vector; p‐CREB Y134F , CREB Y134F ‐GFP‐expressing vector. E FST expression and FLT3 /ITD signaling were detected by Western blotting in Ba/F3‐parental (P in short) and Ba/F3‐ FLT3 /ITD (ITD in short) cells. F–H Phospho‐flow analysis of p‐CREB in Ba/F3‐parental, Ba/F3‐ FLT3 /ITD, and Ba/F3‐ FLT3 /ITD cells treated with FLT3 inhibitor quizartinib (Qui in short). Isotype antibody was used as control to calculate the mean fluorescence intensity (MFI) ratio (F, G). The transcription and expression of Fst were detected by RT–qPCR after quizartinib treatment (10 nM) in Ba/F3‐ FLT3 /ITD cells for 1 day (H). I–K The expression of FST and phosphorylation of CREB were detected by Western blotting (I and K) and phospho‐flow analysis (J) in MOLM‐13 (I) and Ba/F3‐ FLT3 /ITD (K) cells treated with quizartinib and BRD7389 for 1 day, respectively. L RSK expression and FST expression were detected by Western blotting after p90RSK knockout by CRISPR/Cas9 in MOLM‐13 cells. M The phosphorylation of CREB and FST expression was detected by Western blotting in Ba/F3‐ FLT3 /ITD cells treated with CREB inhibitor 666‐15 for 1 day. ^: non‐specific staining of p‐ATF1 protein due to the conserved motif. N CREB expression and FST expression were detected by Western blotting after CREB knockout by CRISPR/Cas9 in MOLM‐13 cells. O The growth of Ba/F3‐parental (with IL‐3), Ba/F3‐ FLT3 /ITD (without IL‐3), and Ba/F3‐ FLT3 /ITD (with IL‐3) cells was measured after 3 days treatment of CREB inhibitor 666‐15 in vitro . P The rescue effect of CREB inhibitor 666‐15 on FLT3 /ITD‐induced dorsalization and axis duplication in zebrafish embryos at 1 dpf. Data information: In (C, D, G, H, J, and O), the experiments were performed in triplicates, and the data were presented as mean ± SEM. ** P < 0.01 and *** P < 0.001 (Student's t ‐test). Source data are available online for this figure.

Article Snippet: MOLM‐13 cell line was infected by pCW‐Cas9, doxycycline‐inducible lentiviral expressing Cas9 (Addgene plasmid #50661), and positively infected cells were selected by 2 μg/ml puromycin. sgRNAs ( ) targeting p90RSK and CREB were cloned into pLKO5.sgRNA.EFS.tRFP657 (Addgene plasmid #57824). sgRNA lentivirus was prepared and used to infect inducible Cas9‐expressing human AML cell lines.

Techniques: In Silico, Binding Assay, ChIP-qPCR, Positive Control, Negative Control, Luciferase, Plasmid Preparation, Expressing, Western Blot, Control, Fluorescence, Quantitative RT-PCR, Phospho-proteomics, Knock-Out, CRISPR, Staining, In Vitro

Journal: EMBO Molecular Medicine

Article Title: Follistatin is a novel therapeutic target and biomarker in FLT 3/ ITD acute myeloid leukemia

doi: 10.15252/emmm.201910895

Figure Lengend Snippet: A, B The morphology and clonogenicity of MOLM‐13 after FST knockout by CRISPR/Cas9 in vitro . Scale bar = 10 μm. C, D The engraftment of MOLM‐13 after FST knockout was detected by flow cytometry of human CD45‐ and mouse CD45.1‐positive cells in recipient mouse BM aspiration at week 2 post‐transplantation. E The effect of FST knockout on the survival of NSG mice engrafted with MOLM‐13 cells. Cas9: Cas9 only (10 mice); sgRNA#3: Cas9 + sgRNA#3 (10 mice); sgRNA#4: Cas9 + sgRNA#4 (8 mice). F The knockdown efficiency of different FST ‐specific antisense oligos (ASOs) in MOLM‐13 cells was detected by RT–qPCR after 3 days of treatment in vitro . The knockdown and RT–qPCR experiments were performed in triplicates. G MOLM‐13 cell growth was measured after 3 days of treatment of FST ‐ASO in vitro . The ASO treatment experiments were performed in triplicates. H Intraperitoneal injection of FST ‐ASO (10 mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM‐13‐engrafted NSG mice. The random sequence was used for negative control (Neg‐ASO, 6 mice). Data information: In (B, D, F, and G), data were presented as mean ± SEM. * P < 0.05 and ** P < 0.01 (Student's t ‐test). In (E and H), survival curves were analyzed by log‐rank test. * P < 0.05.

Article Snippet: MOLM‐13 cell line was infected by pCW‐Cas9, doxycycline‐inducible lentiviral expressing Cas9 (Addgene plasmid #50661), and positively infected cells were selected by 2 μg/ml puromycin. sgRNAs ( ) targeting p90RSK and CREB were cloned into pLKO5.sgRNA.EFS.tRFP657 (Addgene plasmid #57824). sgRNA lentivirus was prepared and used to infect inducible Cas9‐expressing human AML cell lines.

Techniques: Knock-Out, CRISPR, In Vitro, Flow Cytometry, Transplantation Assay, Knockdown, Quantitative RT-PCR, Injection, Sequencing, Negative Control

Journal: eLife

Article Title: The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure

doi: 10.7554/eLife.61797

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , LentiV_Cas9_puro (plasmid) , Addgene , RRID: Addgene_108100 , Lentiviral expression of cDNA with puromycin resistance gene – used for making RPE-1 cas9 puro (generous gift from Jason Sheltzer, CSHL).

Techniques: Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Cloning, CRISPR, Construct, Retroviral, Transfection, Transduction, Sequencing, Amplification, Flow Cytometry, Software, Genome Wide, Knock-Out, Staining

Journal: Hepatology Communications

Article Title: Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

doi: 10.1002/hep4.2014

Figure Lengend Snippet: Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 targets covalently closed circular DNA (cccDNA) and exerts an antiviral effect accompanied by a significant reduction in cccDNA in HBV‐infected hepatoma cells. (A) Western blot of Cas9 and b‐actin in HepG2‐hNTCP‐C4‐iCas9 cells 1 day after doxycycline (DOX) treatment. (B) Representative images of immunofluorescent staining of hepatitis B core (HBc) 13 days after hepatitis B virus (HBV) inoculation. (C–F) HepG2‐hNTCP‐C4‐iCas9 cells lentivirally transduced with the HBV guide RNA (gRNA) or NC gRNA vector were inoculated with HBV (10,000 GEq/cell). (C) Experimental protocol. (D) Intracellular cccDNA levels in HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (10,000 GEq/cell) (n = 4, ** p < 0.01). (E) Indel ratio in the cccDNA of HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (n = 4, * p < 0.05). (F) Intracellular pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (10,000 GEq/cell) (n = 4, ** p < 0.01). DAPI, 4′,6‐diamidino‐2‐phenylindole. NC, negative control.

Article Snippet: HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells, [ ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.

Techniques: CRISPR, Infection, Western Blot, Staining, Virus, Transduction, Plasmid Preparation, Negative Control

Journal: Hepatology Communications

Article Title: Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

doi: 10.1002/hep4.2014

Figure Lengend Snippet: HBV‐CRISPR exerts an antiviral effect accompanied by a significant reduction in cccDNA in HBV‐infected primary human hepatocytes (PHHs). PHHs isolated from humanized liver chimeric mice were lentivirally transduced with tandem Cas9 gRNA and HBV gRNA (or NC gRNA)–expressing vectors (10 multiplicity of infection [MOI]) 19 days after HBV inoculation (500 GEq/cell). (A) Experimental protocol. (B) cccDNA levels, intracellular pgRNA levels, and supernatant HBV DNA, HBs antigen, and HBe antigen levels in PHHs 35 days after HBV inoculation (n = 4; * p < 0.05, ** p < 0.01).

Article Snippet: HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells, [ ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.

Techniques: CRISPR, Infection, Isolation, Transduction, Expressing

Journal: Hepatology Communications

Article Title: Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

doi: 10.1002/hep4.2014

Figure Lengend Snippet: Olaparib enhances the antiviral effect of HBV‐CRISPR in HBV‐infected hepatoma cells and PHHs. (A–C) HepG2‐hNTCP‐C4‐iCas9 cells transduced with HBV gRNA were treated with or without olaparib (1 μM) 10 days after HBV inoculation (10,000 GEq/cell). (A) Experimental protocol. (B) Intracellular cccDNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation. (C) pgRNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation. (D–F) HepG2‐hNTCP‐C4‐iCas9 cells transduced with HBV gRNA were treated with DOX 4 days after HBV inoculation (10,000 GEq/cell) and then with olaparib (1 μM) or vehicle 10 days after HBV inoculation. (D) Experimental protocol. (E) cccDNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (n = 4; * p < 0.05). (F) Intracellular pgRNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (n = 4; ** p < 0.01). (G–I) PHHs isolated from humanized liver chimeric mice were lentivirally transduced with tandem Cas9‐expressing and HBV gRNA–expressing vectors (10 MOI) 19 days after HBV inoculation (500 GEq/cell) and were then treated with olaparib (1 μM) or vehicle. (G) Experimental protocol. (H) cccDNA levels in PHHs 35 days after HBV inoculation (n = 4; * p < 0.05). (I) Intracellular pgRNA levels in PHHs 35 days after HBV inoculation (n = 4, * p < 0.05).

Article Snippet: HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells, [ ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.

Techniques: CRISPR, Infection, Transduction, Isolation, Expressing

Journal: Cell metabolism

Article Title: Cytosolic Aspartate Availability Determines Cell Survival When Glutamine Is Limiting

doi: 10.1016/j.cmet.2018.07.021

Figure Lengend Snippet: Key Resources Table

Article Snippet: pLentiCRISPRv2 (lentiviral Cas9 expressing vector) , Laboratory of Dr. Feng Zhang , Addgene Cat #52961.

Techniques: Recombinant, shRNA, Expressing, Plasmid Preparation, Software, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9.

doi: 10.1073/pnas.2103532119

Figure Lengend Snippet: Fig. 3. CINDELA with CRISPR-Cas9. (A) CINDELA-induced cancer cell death introduced by the SpCas9 RNP complex with 30 gRNAs targeting U2OS- specific InDels. (Scale bar, 300 μm.) (B) CINDELA-induced cell death intro- duced by the lentivirus-delivered SpCas9. We tested by using multiple tar- get gRNAs in conjunction with cell type–specific gRNAs (23 sgRNAs for HCT-116 and 21 sgRNAs for U2OS). The relative cell viability and the pro- portion of apoptotic cells confirmed the specific cell death by CINDELA both on multiple target gRNAs and cell type–specific InDel targeting gRNAs. (C) CINDELA-induced cell death introduced by the AAV-delivered SaCas9. When we pooled AAV with 30 different gRNAs targeting U2OS- specific InDels and SaCas9 and delivered it to U2OS cells. Compared with the control (with no gRNA), a significant number of cells died.

Article Snippet: The lentiviral plasmid expressing CRISPR/ SpCas9 under the EF1-α promoter (Addgene, no. 52962) was used to produce lentiviral production as described previously (22).

Techniques: CRISPR, Control

Journal: Cell Death & Disease

Article Title: MEK5/ERK5 inhibition sensitizes NRAS -mutant melanoma to MAPK-targeted therapy by preventing Cyclin D/CDK4-mediated G1/S progression

doi: 10.1038/s41419-025-08036-7

Figure Lengend Snippet: A : Representative immunoblots of n = 3 experiments done using total lysates from BLM wild-type (Wt), BLM empty vector (EV)-infected, or two different CRISPR/ Cas9 -mediated MEK5 k.o. single cell clones (SCC) of BLM treated with diluent (ctrl) or 25 nM Tram for 14 days, showing expression of selected cell cycle proteins. ERK5 phosphorylation and DUSP4 suppression confirm Tram functionality. Successful MEK5 gene disruption was analysed by MEK5 immunoblot and functionally evaluated by immunoblotting for ERK5, which confirmed absent ERK5 autophosphorylation. Tubulin served as loading control. B : Representative cell cycle profiles of n = 2 experiments, as determined by flow cytometric analysis of the indicated PI-stained conditions with percentages of S-phase cells indicated. C : Cell doubling time analysis representative of n = 2 experiments with running times of 2–5 weeks performed with Wt BLM or the indicated MEK5 k.o. BLM SCCs cultured in absence or presence of Tram. The shown experiment run over a period of five weeks.

Article Snippet: BLM, lentiCRISPR_zeo, a derivative of the lentiviral dual gRNA/ Cas9 expression vector lentiCRISPRv2-Blast (Addgene_83480) replacing the blasticidin R gene by a zeocin R gene was used.

Techniques: Western Blot, Plasmid Preparation, Infection, CRISPR, Clone Assay, Expressing, Phospho-proteomics, Disruption, Control, Staining, Cell Culture

Journal: Nature Communications

Article Title: Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer

doi: 10.1038/s41467-020-18013-7

Figure Lengend Snippet: AR protein levels in ( a ) LNCaP cells treated with EPZ followed by quantitation ( n = 5). b AR protein in VCaP and C42B cells after EPZ treatment. c shControl or shDOT1L transfected LNCaP and C42B cells. d PDX organoids treated with EPZ. e AR protein levels in EV or DOT1L overexpressing LNCaP cells. f Proliferation assay of LNCaP cells with and without DOT1L expression in charcoal stripped media measured using MTS assays for 4 days. g AR western analysis after 50 μg/ml Cycloheximide treatment in LNCaP cells treated with Vehicle or EPZ for 8 days (left). Representative experiment shown. Quantitation of AR protein levels from 3 independent experiments (right). h PSA and AR western blot analysis in LNCaP treated with Vehicle and EPZ for 8 days. Representative image shown. i Percentage of RFP + GFP + cells counted by Flow cytometry after 8 days of Vehicle or EPZ treatment in LNCaP cells transfected with ARE-GFP reporter construct. j GSEA plot of Nelson_Response_to_Androgen geneset enriched in LNCaP cells treated with 1 μM EPZ for 8 days compared to Vehicle treatment. mRNA expression of 6 AR target genes measured by qRT-PCR in LNCaP cells after 8 days of ( k ) 1 μM EPZ treatment and ( l ) transduction with shDOT1L or shControl lentivirus. m Relative enrichment of AR at 4 target genes measured by ChIP followed by qPCR in LNCaP cells treated with Vehicle or 1 μM EPZ for 8 days. n ChIP-seq plots of H3K79me2 at two AR target genes in LNCaP cells treated with Vehicle or 1 μM EPZ. Statistical tests: p value determined by two-tailed t test ( a , f – i , k – m ) corrected for multiple comparisons in ( k – m ). n = 3 independent experiments ( b , c , f , h – m ). FDR < 25% ( j ). Error bars represent S.E.M. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To stably express CAS9 in LNCaP and PC3 cells, we generated a CAS9 (Streptococcus pyogenes CRISPR-Cas) expressing lentivirus (Addgene 65655) from 293T cells.

Techniques: Quantitation Assay, Transfection, Proliferation Assay, Expressing, Western Blot, Flow Cytometry, Construct, Quantitative RT-PCR, Transduction, ChIP-sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer

doi: 10.1038/s41467-020-18013-7

Figure Lengend Snippet: a Comparison of leading-edge genes from Nelson_Response_To_Androgen dataset in the present study ( n = 35) and Barfeld study ( n = 40). b GSEA plots of two MYC related datasets enriched in either Vehicle treated (left) or EPZ-treated (right) LNCaP cells. Cells were treated with 1 μM Vehicle or EPZ for 8 days prior to microarray analysis ( n = 3). c mRNA expression of MYC in (left) LNCaP cells treated with Vehicle or 1 μM EPZ and LNCaP cells transduced with shControl or shDOT1L lentivirus; (right) PC3 cells treated with Vehicle or 1 μM EPZ for 8 days. d Positive correlation between DOT1L and MYC expression in the MSKCC dataset ( n = 150). Data were obtained from cbioportal.org . e Western blot analysis of MYC protein in LNCaP and PC3 cells treated with Vehicle or EPZ 1 μM for 8 days. Representative images shown. Western blot analysis of MYC protein in ( f ) PDX organoids treated with EPZ for 8 days, g LNCaP cells treated with EPZ5676 for 8 days, h LNCaP, C42B, and 22rv1 cells with DOT1L knockdown and i LNCaP, C42B, and 22rv1 cells with DOT1L overexpression. Representative images shown. j Western blot analysis and quantitation of MYC protein after treatment with 50 ug/ml Cycloheximide in LNCaP cells treated with vehicle or 1 μM EPZ for 8 days. k Western blot analysis of MYC protein after treatment with 10 μM MG-132 in LNCaP cells treated with vehicle or 1 μM EPZ for 8 days. Representative images shown. Statistical tests: p value determined by Hypergeometric test ( a ), Spearman’s rank correlation ( d ), and two-tailed Student’s t test ( c , j ). Error bars represent S.E.M. n = 3 independent experiments ( c , e – k ). ** p < 0.01; **** p < 0.0001.

Article Snippet: To stably express CAS9 in LNCaP and PC3 cells, we generated a CAS9 (Streptococcus pyogenes CRISPR-Cas) expressing lentivirus (Addgene 65655) from 293T cells.

Techniques: Comparison, Microarray, Expressing, Transduction, Western Blot, Knockdown, Over Expression, Quantitation Assay, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Expression of markers of metabolic compartmentalization in non-small cell lung cancer (NSCLC). Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patient samples were stained for MCT4, MCT1 and TOMM20 by immunohistochemistry. For each marker, representative images were taken from tumor tissue and adjacent normal lung tissue within the same sample. (A, B) MCT4 expression in the tumor stroma of LUAD (A) and LUSC (B) and their corresponding adjacent normal lung. (C, D) MCT1 expression in carcinoma cells in LUAD (C) and LUSC (D) and their corresponding adjacent normal lung. (E, F) TOMM20 expression in carcinoma cells in LUAD (E) and LUSC (F) and their corresponding adjacent normal lung. Images were taken at 20X. (S, stroma; C, carcinoma cells).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Staining, Immunohistochemistry, Marker

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Expression of markers of metabolic compartmentalization in co-cultures of ADT carcinoma cells and fibroblasts. Human ADT carcinoma cell lines A549, HCC827, H226, H23, SCC9 and SCC25 were co-cultured with BJ1 fibroblasts for 4 days. Monocultures of carcinoma cells and BJ1 fibroblasts were maintained in parallel as controls. (A–D) Confocal imaging of MCT4 in monocultures of BJ1 and in co-cultures of BJ1 with the NSCLC cells HCC827, H226 and H23 (A) and HNSCC cells SCC9 and SCC25 (C) , and their respective quantification of MCT4 staining in BJ1 (B, D) . (E, F) . Confocal imaging of IDH3α in monocultures of BJ1 and in co-cultures of BJ1 with A549 and HCC827 (E) , and quantification of IDH3α staining in BJ1 (F) . (G–L) Confocal imaging of TOMM20 in monocultures and co-cultures of A549 (G) , HCC827 (I) , and SCC9 (K) with BJ1, and quantification of TOMM20 staining in A549 (H) , HCC827 (J) and SCC9 (L) . Confocal microscopy images were acquired at the 40X magnification, with 1.5x zoom in panel (E) . For all images, MCT4, IDH3α and TOMM20 staining are shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). For all markers, red staining was quantified using ImageJ. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Cell Culture, Imaging, Staining, Confocal Microscopy

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: PEPCK-M expression in glycolytic fibroblasts. BJ1 and WT or MCT4-KO MEF were co-cultured with ADT carcinoma cells. Monocultures of BJ1 and MEF were maintained in parallel as controls. (A–D) Confocal imaging of PEPCK-M in monocultures and in co-cultures of BJ1 with A549 (A) and SCC25 (C) , and their respective quantification of PEPCK-M staining in BJ1 (B, D) . (E, F) Confocal imaging of PEPCK-M in monocultures of WT and MCT4-KO MEF (E) and quantification of PEPCK-M staining (F) . (G–L) Confocal imaging of PEPCK-M in WT and MCT4-KO MEF in co-culture with A549 (G) , SCC9 (I) and SCC25 (K) , and their respective quantification of PEPCK-M staining in MEF (H, J, L) . Confocal microscopy images were acquired at the 40X magnification. For all images, PEPCK-M staining is shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). PEPCK-M staining was quantified using ImageJ. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Cell Culture, Imaging, Staining, Co-Culture Assay, Confocal Microscopy

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of HIF-1α inhibition on fibroblast metabolism and carcinoma cell aggressiveness. Co-cultures of ADT carcinoma cells and BJ1 were treated with the HIF-1α inhibitor BAY 87-2243 or vehicle control for 48 hours. (A–D) Confocal imaging of IDH3α in co-cultures of BJ1 with A549 (A) and HCC827 (C) treated or untreated with BAY 87-2243, and their respective quantification of IDH3α staining in BJ1 (B, D) . (E–H) Confocal imaging of MCT4 in co-cultures of BJ1 with A549 (E) and HCC827 (G) treated or untreated with BAY 87-2243, and their respective quantification of MCT4 staining in BJ1 fibroblasts (F, H) . Confocal microscopy images were acquired at the 40X magnification, with 1.5x zoom in panels (A, C) . For all images, IDH3α and MCT4 staining are shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). IDH3α and MCT4 staining were quantified using ImageJ. (I–L) Flow cytometry assessment of percentage of carcinoma cells undergoing apoptosis (AnnV staining) and cell death (PI staining) in co-cultured A549 (I, J) and HCC827 (K, L) untreated or treated with BAY 87-2243 with representative flow cytometry plots showing gating strategy for AnnV (APC) and PI (PE) staining. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Inhibition, Control, Imaging, Staining, Confocal Microscopy, Flow Cytometry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of ROS neutralization and MCT4 downregulation on fibroblast metabolism and ADT carcinoma cell aggressiveness. Co-cultures of ADT carcinoma cells with BJ1 were treated with N-acetyl cysteine (NAC) for 24 hours for proliferation assessments, and for 48 hours for confocal imaging and apoptosis assessment. (A–D) Confocal imaging of MCT4 in co-cultures of BJ1 with A549 (A) and HCC827 (C) treated or untreated with NAC, and respective quantification of MCT4 staining in BJ1 (B, D) . Confocal microscopy images were acquired at the 40X magnification. For all images, MCT4 staining is shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). MCT4 staining was quantified using ImageJ. (E, F) Flow cytometry quantification of percentage of A549 cells in the DNA synthesis phase, measured by 5-ethynyl-2’-deoxyuridine (EdU) incorporation (E) , and gating strategy for EdU (Pacific Blue) and FxCycle (APC) staining (F) . (G–J) Flow cytometry assessment of percentage of carcinoma cells undergoing apoptosis (AnnV staining) and cell death (PI staining) in co-cultured A549 (G, H) and HCC827 (I, J) cells untreated or treated with NAC with representative flow cytometry plots showing gating strategy for AnnV (APC) and PI (PE) staining. (K–M) ADT carcinoma cells were co-cultured with mCherry-tagged BJ1 with downregulated MCT4 expression (BJ1-sgMCT4) or their control counterparts (BJ1-sgCTRL) and apoptosis (AnnV staining) and cell death (DAPI staining) was assessed in A549 (K) , HCC827 (L) and H520 (M) by flow cytometry. (N) GFP-tagged A549 were monocultured or co-cultured with WT or MCT4-KO MEFs and proliferation rates in A549 were assessed by flow cytometric analysis of EdU incorporation. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm). ns, not significant.

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Neutralization, Imaging, Staining, Confocal Microscopy, Flow Cytometry, DNA Synthesis, Cell Culture, Expressing, Control

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of fibroblast MCT4 on tumor growth. (A–J) Tumor xenografts were generated by co-injecting human ADT carcinoma cells with fibroblasts expressing or lacking MCT4 into nude mice. (A) Tumor volume and (B) tumor weight of HCC827 + BJ1-sgCTRL/sgMCT4 xenografts harvested at 2.5 weeks post-implantation. (C, D) Immunohistochemical assessment of mCherry (to detect BJ1) and MCT4 expression in HCC827 + BJ1-sgCTRL/sgMCT4 tumors, and image acquisition at 40x (C) and 60x (D) . In panel (D) the area of carcinoma cells, mouse stroma, and injected mCherry-expressing BJ1 (*) are marked. (E) Tumor volume and (F) tumor weight of HCC827 + WT/MCT4-KO MEF xenografts harvested at 4 weeks post-implantation. (G) Tumor volume and (H) tumor weight of H520 + BJ1-sgCTRL/sgMCT4 xenografts harvested at 2 months post-implantation. (I) Tumor volume and (J) tumor weight of SCC25 + BJ1-sgCTRL/sgMCT4 xenografts harvested at 6 weeks post-implantation. (K–N) Syngeneic tumors were generated by co-injecting C57BL/6 carcinoma cells with WT or MCT4-KO MEFs into WT or MCT4-KO C57BL/6 mice. (K) Tumor volume and (L) tumor weight of MOC1 + WT/MCT4-KO MEF tumors harvested at 7 weeks post-implantation. (M) Tumor volume and (N) tumor weight of MOC2 + WT/MCT4-KO MEF tumors harvested at 3 weeks post-implantation. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Generated, Expressing, Immunohistochemical staining, Injection

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of fibroblast MCT4 on tumor metabolism. The metabolic markers MCT4, GLUT1, MCT1 and TOMM20 were assessed by immunohistochemical staining in tumor xenografts generated from the co-injection of HCC827 with BJ1-sgCTRL or BJ1-sgMCT4. Representative IHC images of MCT4 (A) , GLUT1 (D) , MCT1 (G) and TOMM20 (J) staining. Membranous staining of MCT4 (B, C) , GLUT1 (E, F) and MCT1 (H, I) in HCC827 cells was identified with the ImmunoMembrane software and quantified with ImageJ. The ImmunoMembrane software detects and labels strong membranous staining in red and weak staining in green. Only staining labeled in red was quantified by ImageJ as the area in pixels of the total image covered by the red labeling. Mitochondrial staining of TOMM20 on HCC827 cells was quantified by Aperio (K) . Representative images for each group are shown. All IHC images were acquired at 40X, except for MCT4 quantification in panel (B) that is 20X. Student’s t-test was used for statistical analyses (*p<0.05).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Immunohistochemical staining, Staining, Generated, Injection, Software, Labeling

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Metabolic compartmentalization in ADT cancers and interventions to modulate fibroblast metabolism and carcinoma cell aggressiveness. (A) We have demonstrated that both in patients and in experimental models of ADT cancers, CAFs are glycolytic, whereas carcinoma cells are mitochondria-rich and have OXPHOS metabolism. We have shown that CAFs have increased levels of ROS. ROS are a mean of communication between metabolic compartments and known inducers of HIF-1α stabilization in CAFs. HIF-1α drives glycolysis and transcriptionally regulates MCT4 and PEPCK-M. We have shown that CAFs have upregulation of MCT4 and PEPCK-M, and that PEPCK-M expression may also be regulated by MCT4. MCT4 is located in the plasma membrane and is the main exporter of glycolysis-derived lactate. Loss of IDH3α is also a driver of HIF-1α stabilization. We have shown that CAFs have downregulation of IDH3α expression and that elevated levels of HIF-1α may in turn maintain the low expression of IDH3α. Both upregulation of MCT4 and loss of IDH3α are markers of glycolysis in CAFs, and we define PEPCK-M as another possible marker of glycolytic reprogramming in CAFs. We have demonstrated that CAF glycolytic reprogramming can be targeted using a HIF-1α inhibitor (BAY 87-2243) and an antioxidant (NAC). In the carcinoma cell compartment, we have demonstrated that the presence of glycolytic CAFs induces ROS, MCT1 and TOMM20 expression and drives carcinoma cell aggressiveness, as seen by decreased cell death and increased proliferation, and tumor growth. In bold are the elements studied in our report. Black arrows indicate previously described pathways. Green arrows indicate the novel findings reported here. Red inhibitor arrows indicate NAC and BAY 87-2243 targets. (B) We have modulated CAF glycolytic metabolism and ADT carcinoma cell aggressiveness by means of pharmacological and genetic interventions. The HIF-1α inhibitor BAY 87-2243 rescued IDH3α expression in CAFs and induced carcinoma cell apoptosis. The antioxidant NAC reduced ROS levels and MCT4 expression in CAFs, and in carcinoma cells it reduced ROS levels, induced apoptosis and decreased proliferation. Genetic knock-down (KD) or knock-out (KO) of MCT4 in CAFs increased apoptosis and decreased proliferation in carcinoma cells, and reduced growth and the expression of MCT1, TOMM20, MCT4 and GLUT1 in tumors. Yellow boxes indicate effects in vitro . Red boxes indicate effects in vivo . CAF, Cancer-associated fibroblast; ROS, reactive oxygen species; HIF-1α, hypoxia inducible factor 1 alpha; MCT4, monocarboxylate transporter 4; IDH3α, isocitrate dehydrogenase 3 alpha; PEPCK-M, mitochondrial phosphoenolpyruvate carboxykinase; NAC, N-acetyl cysteine; MCT1, monocarboxylate transporter 1; TOMM20, translocase of the outer mitochondrial membrane 20. Created with BioRender.com .

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Clinical Proteomics, Membrane, Derivative Assay, Marker, Knockdown, Knock-Out, In Vitro, In Vivo