Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Adaptable expression ratios of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 3xHA -P2A-K n -MCT12 2xmyc construct. (B) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (C) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 5). Data are represented as mean ± SEM. (D) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (E) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 4). Data are represented as mean ± SEM. See also .

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Western Blot, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Relative cell surface expression of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 eGFP -P2A-K 0 -MCT12 mCherry (a) and MCT12 mCherry -P2A-K 0 -CRT1 eGFP construct (b). (B) Ratio of mCherry and eGFP expression in eGFP-P2A-K 0 -mCherry (K 0 , as a control), CRT1 eGFP -P2A-K 0 -MCT12 mCherry , and MCT12 mCherry -P2A-K 0 -CRT1 eGFP constructs as determined by flow cytometry ( n = 6). Data are represented as mean ± SEM. (C) Live-cell confocal microscopy of HEK293 SLC6A8 −/− cells expressing CRT1 eGFP -P2A-K 0 -MCT12 mCherry and MCT12 mCherry -P2A-K 0 -CRT1 eGFP . The plasma membrane was stained with CellMask Deep Red. Scale bars, 20 μm. (D) Analysis of cell surface expression of CRT1 eGFP and MCT12 mCherry . n = 30 (CRT1 eGFP -P2A-K 0 -MCT12 mCherry ); n = 28 (MCT12 mCherry -P2A-K 0 -CRT1 eGFP ).

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Control, Flow Cytometry, Confocal, Microscopy, Clinical Proteomics, Membrane, Staining, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet:

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Cell-Signaling, Virus, Recombinant, Clinical Proteomics, Membrane, Staining, Cloning, Software

Journal: iScience

Article Title: Sulfonylurea receptor 1 deficiency alleviates acute lung injury by activating PARP1 in epithelial cells

doi: 10.1016/j.isci.2026.117627

Figure Lengend Snippet: SUR1 protein levels were elevated in the bronchoalveolar lavage fluid (BALF), but not the serum, of sepsis-related ALI patients and CLP-induced ALI mouse models (A) Dot blot analysis of BALF and serum collected from sepsis-related ALI patients or controls. NC (negative control), PBS; PC (positive control), whole-cell protein lysates of HEK293T cells. Control, patients who underwent abdominal surgery without ALI or bacterial infection; ALI, sepsis-associated ALI. Red circle, site of PBS drops. (B) Cytokine levels in BALF and serum of patients determined by a cytokine screening assay. n = 3 for control; n = 9 for ALI. (C) Dot blot analysis of BALF and serum from CLP-induced ALI mice ( n = 6). (D–F) Western blot and RT-qPCR analyses of lung tissue samples from CLP-induced ALI mice. Data are presented as mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significance. Data in (A, B, C, and F) were analyzed using Welch’s t test, and data in (E) were analyzed using an unpaired two-tailed Student’s t test. Representative images are shown.

Article Snippet: Human embryonic kidney cell line HEK293T , American Type Culture Collection , N/A.

Techniques: Dot Blot, Analysis, Negative Control, Positive Control, Control, Infection, Screening, Western Blot, Quantitative RT-PCR, Tissue, Two Tailed Test

Journal: iScience

Article Title: Sulfonylurea receptor 1 deficiency alleviates acute lung injury by activating PARP1 in epithelial cells

doi: 10.1016/j.isci.2026.117627

Figure Lengend Snippet: SUR1 interacted with PARP1 to suppress DNA damage repair and induce apoptosis of bronchial epithelial cells (A) Co-immunoprecipitation and western blot analyses of HEK293T cells transfected with FLAG-SUR1 plasmids (left) or human bronchial epithlial (HBE) cells (right). (B) Immunofluorescence staining of HBE cells. Scale bars, 25 μm. (C) Homologous recombination reporter assay in U2OS-DRGFP cells transfected with SUR1 siRNA, followed by flow cytometry analysis. (D) Western blot analysis of HBE cells transfected with SUR1 siRNAs with or without 20 ng/mL TNF-α or 50 μg/mL LPS treatment for 1 h. (E) ROS levels in HBE cells with SUR1 knockdown, treated with or without TNF-α 20 ng/mL for 3 h. (F) Apoptosis assay by flow cytometry in HBE cells transfected with SUR1 siRNAs and treated with TNF-α. Data are presented as mean ± SD ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Co-localization in (B) was assessed using Pearson’s correlation coefficient. Data in (C–F) were analyzed using two-way ANOVA followed by Tukey’s multiple-comparisons test. Representative images are shown.

Article Snippet: Human embryonic kidney cell line HEK293T , American Type Culture Collection , N/A.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Immunofluorescence, Staining, Homologous Recombination, Reporter Assay, Flow Cytometry, Analysis, Knockdown, Apoptosis Assay

Journal: iScience

Article Title: Sulfonylurea receptor 1 deficiency alleviates acute lung injury by activating PARP1 in epithelial cells

doi: 10.1016/j.isci.2026.117627

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney cell line HEK293T , American Type Culture Collection , N/A.

Techniques: Cell-Signaling, Recombinant, Transfection, Caspase-3 Activity Assay, Protein Extraction, Knock-Out, Sequencing, Control, Plasmid Preparation, Software, Analysis

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A to C and G ) eGFP reporter assay to assess the silencing efficiency of 3-nt tiled Psp Cas13b crRNAs targeting the breakpoint region of fusion transcripts BCR::ABL1-p190 (A), SNX2::ABL1 (B), SFPQ::ABL1 (C), and BCR::ABL1-p210 (G). Data points in the graphs are averages of mean fluorescence; N = 3. The data are represented in arbitrary units (A.U.). Errors are SEM, and P values of the one-way analysis of variance (ANOVA) test with Dunnett’s multiple comparisons are indicated (95% confidence interval). ( D to F ) RT-PCR assays measuring silencing efficiency of tiled Psp Cas13b crRNAs (3-nt resolution) targeting the breakpoint regions of fusion transcripts (D) BCR::ABL1-p190, (E) SNX2::ABL1, and (F) SFPQ::ABL1; N = 3. Data are normalized means and errors are SEM. Results are analyzed by one-way ANOVA test (Dunnett’s multiple comparisons) with P values indicated (95% confidence interval). ( H ) Representative Western blot analysis to examine the expression level of BCR::ABL1-p190 protein in HEK 293T cells expressing tiled crRNAs targeting BCR::ABL1-p190 transcripts 24 hours posttransfection, with quantification from three independent experiments; N = 3. n.s., not significant. ( I ) Schematic of BCR::ABL1-dependent phosphorylation of ERK and STAT proteins, and inhibition of BCR::ABL1 oncogenic activity with imatinib, a potent competitor of adenosine 5′-triphosphate (ATP) binding to ABL kinase. ( J ) Representative Western blot analysis examining the suppression of BCR::ABL1 expression and the subsequent inhibition of STAT5 and ERK phosphorylation in HEK 293T cells expressing BCR::ABL1-p190, Psp Cas13b, and either NT or crRNA targeting the BCR::ABL1 24 hours posttransfection. HEK 293T cells expressing BCR::ABL1-p190 and Psp Cas13b treated with 1 μM imatinib for 4 hours were used as a positive control. Parental cells are HEK 293T cells transfected with Psp Cas13b, NT, and a control plasmid; N = 3. N is the number of independent biological experiments.

Article Snippet: The HEK 293T cell line [American Type Culture Collection (ATCC), CRL-3216] was cultured in Dulbecco’s modified Eagle’s medium high-glucose medium (Thermo Fisher Scientific, 11965092) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147), Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220), and 2 mM GlutaMAX (Thermo Fisher Scientific, A1286001).

Techniques: Reporter Assay, Fluorescence, Analysis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Phospho-proteomics, Inhibition, Activity Assay, Binding Assay, Positive Control, Transfection, Control, Plasmid Preparation

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A and B ) Comprehensive analysis of spacer-target interaction examining specificity and mismatch tolerance of Psp Cas13b crRNAs targeting the breakpoint region of BCR::ABL1-p190 transcript. The nucleotides in red highlight various mismatch positions in the spacer sequence. Data points in the graph are averages of mean fluorescence from four representative fields of views per condition imaged; N = 4. The data are represented in A.U. Errors are SEM, and P values of the one-way ANOVA test with Dunnett’s multiple comparisons are indicated (95% confidence interval). ( C ) Western blot analysis examining the expression level of BCR::ABL1-p190 protein and phosphorylation status of STAT5 and ERK in HEK 293T cells expressing crRNAs with various mismatches 24 hours posttransfection; N = 3 (uncropped blots and protein quantifications are available in the Supplementary Materials.). ( D to F ) Three-color fluorescence-based reporter assays to assess the specificity of crRNA targeting the breakpoint region of BCR::ABL1 in HEK 293T cells 48 hours posttransfection. The schematics show (D) BCR::ABL1-mCherry mRNA, (E) ABL1-eGFP mRNA, and (F) BCR-TagBFP mRNA and their interaction with crBCR, crBCR::ABL1, and crABL1 crRNAs through full, partial, or no spacer-target base pairing. The quantifications of the silencing of (E) BCR::ABL1-mCherry, (F) ABL1-eGFP, and ( G ) BCR-TagBFP. Data points are averages of normalized mean fluorescence from four representative fields of view per condition imaged. The data are represented in A.U. Errors are SEM, and P values of the one-way ANOVA test with Dunnett’s multiple comparisons are indicated (95% confidence interval); N = 3. The right panels show representative Western blot analyses to examine the expression levels of ΔBCR::ABL, ΔABL1, and ΔBCR proteins from data in (D) to (F); N = 3 (see uncropped blots in the Supplementary Materials). N represents the number of biological replicates. See Dryad for source data.

Article Snippet: The HEK 293T cell line [American Type Culture Collection (ATCC), CRL-3216] was cultured in Dulbecco’s modified Eagle’s medium high-glucose medium (Thermo Fisher Scientific, 11965092) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147), Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220), and 2 mM GlutaMAX (Thermo Fisher Scientific, A1286001).

Techniques: Analysis, Sequencing, Fluorescence, Western Blot, Expressing, Phospho-proteomics

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A and B ) Mean-difference plots showing the log 2 fold change (FC) and average abundance of transcripts. The full-length BCR::ABL1 sequence (A) or a 180-nt-long flanking Psp Cas13b binding site at the breakpoint (B) was included in the reference genome for read alignment and quantification, respectively; N = 3. The histograms depict counts per million (CPM) of reads mapped to the full-length BCR::ABL1 transcript (A) or the BCR::ABL1 breakpoint (B). ( C ) Representative Arriba fusion plots showing read alignments against the BCR::ABL1 transcript. Sashimi plots (bottom) depict the frequency and positions of reads with aberrant junctions. The breakpoint region is highlighted with a dashline box. ( D ) Counts per million (top) and relative frequency (bottom) of aberrant reads exclusively mapped to BCR::ABL1 breakpoint in HEK 293T cells. ( E ) Schematic showing the design of RT-qPCR primers binding different regions on BCR::ABL1-p190-IRES-eGFP transcript (top). RT-qPCR assays (bottom) to measure BCR::ABL1-p190 and eGFP transcript abundance; N = 3. Data are normalized means, and errors are SEM. Results are analyzed by unpaired two-tailed Student’s t test (95% confidence interval). ( F to I ) Detection of BCR::ABL1 aberrant transcripts containing internal deletions in HEK 293T cells obtained using long-read direct RNA-seq (Oxford Nanopore); N = 3. (F) Counts per million of BCR-ABL1 reads. Results are analyzed by unpaired two-tailed Student’s t test (95% confidence interval). (G) Frequency and positions of internal deletions. (H) Length of deletions in BCR::ABL1 aberrant transcripts. Dashed lines indicate the median. (I) Proportions of in-frame and out-of-frame aberrant reads. ( J ) Arriba fusion plots showing the read alignments against PAX3::FOXO1 gene fusion in Rh30 cells following RfxCas13d-mediated cleavage using NT (control), olfactory receptor–targeting (OR; control), and PAX3::FOXO1-targeting crRNA1/crRNA2. The Sashimi plots at the bottom illustrate aberrant read junctions; N = 4. ( K ) Relative frequency of aberrant reads exclusively mapped to PAX3::FOXO1 breakpoint; N = 4. N represents the number of biological replicates.

Article Snippet: The HEK 293T cell line [American Type Culture Collection (ATCC), CRL-3216] was cultured in Dulbecco’s modified Eagle’s medium high-glucose medium (Thermo Fisher Scientific, 11965092) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147), Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220), and 2 mM GlutaMAX (Thermo Fisher Scientific, A1286001).

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Control, Olfactory

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A to C ) Schematic (top) of Psp Cas13b crRNAs targeting the breakpoint sequence of (A) RUNX1-RUNX1T1, (B) NPM-ALK, and (C) SS18-SSX1/2 mRNAs. The bar graphs (middle) quantify the silencing efficiency of crRNAs in HEK 293T cells. Data points in the graph are averages of normalized mean fluorescence from four representative fields of view per experiment imaged; N = 3. The data are represented in A.U. Errors are SEM, and P values of the one-way ANOVA test with Dunnett’s multiple comparisons are indicated (95% confidence interval). Representative Western blot and quantification analysis (bottom) to examine the expression level of (A) RUNX1-RUNX1T1, (B) NPM-ALK, and (C) SS18-SSX1/2 in HEK 293T cells; N = 3. Errors are SEM, and P values of the one-way ANOVA test with Dunnett’s multiple comparisons are indicated (95% confidence interval). N is the number of independent biological experiments. ( D ) Design principles for potent, specific, and personalized silencing of various gene fusion mRNAs with Psp Cas13b nuclease. The left panel shows the rational design of crRNAs targeting the breakpoint of gene fusion transcripts, which enables efficient and specific silencing of these oncogenic drivers, as well as drug-resistant fusion mutants. A fraction of cleaved targets harbor internal deletions near PspCas13b cleavage site. The cleavage disrupts the translation of fusion mRNA. This targeting of the breakpoint strategy avoids the off-targeting of untranslocated gene partners that are expressed in normal tissues. The top right panel shows spacer nucleotide positions that are important for the rational design of potent crRNA ( https://cas13target.azurewebsites.net/ ) . The bottom right panel shows the versatility of Psp Cas13b to silence various undruggable gene fusion transcripts and their oncogenic networks in a personalized manner through precise targeting of fusion’s breakpoint sequence that is exclusively expressed in tumor cells.

Article Snippet: The HEK 293T cell line [American Type Culture Collection (ATCC), CRL-3216] was cultured in Dulbecco’s modified Eagle’s medium high-glucose medium (Thermo Fisher Scientific, 11965092) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147), Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220), and 2 mM GlutaMAX (Thermo Fisher Scientific, A1286001).

Techniques: Sequencing, Fluorescence, Western Blot, Analysis, Expressing