pyogenes cas9  (Cell Signaling Technology Inc)

Journal: Cancer Biology & Therapy

Article Title: Thioredoxin reductase-1 facilitates cellular plasticity and stemness mediated by TGF-β1 in non-small cell lung cancer

doi: 10.1080/15384047.2026.2724630

Figure Lengend Snippet: TXNRD1 potentiates cellular motility and cancer stemness in NSCLC cells. (A) CRISPR/Cas9 sgRNA targeting two sites in the TXNRD1 gene was delivered into A549 cells. Cell sorting of dCas9-GFP-positive cells from flow cytometry of TXNRD1-transfected A549 cells was required to create a single-cell clone. An immunoblot analysis was further performed to validate CRISPRi-TXNRD1 in A549 cells. Western blotting analysis in A549 cells to examine the protein expression of TXNRD1, Cas9, and internal control ( β -actin). (B and C) Knockdown of TXNRD1 by CRISPR/ Cas9 decreased cell migration (B) and invasion (C) in A549 cells. (D–F) Time-lapse analysis of migratory ability by CRISPR knockdown of TXNRD1 in A549 cells, and the culture was traced for 16 h. The indicated group of cells was grown in a culture dish with Matrigel/Collagen coating. The representative wind rose plots of fields per well were taken at 3-minute intervals during 16 h, and the trajectory tracks of the indicated groups covered by A549 cells were plotted with the initial position of each trajectory track during the 16 h of recording. The directional ratios of trajectory per cell (E) and average cell velocity (F) were calculated from time-lapse video microscopy as described in the Materials and Methods. Data represent the mean ± SD ( n = 5). All data statistics were based on * p < 0.05, ** p < 0.01, and *** p < 0.001 calculated by one-way ANOVA with the Brown–Forsythe test. (G) Tumorspheres formation assay in knockdown of TXNRD1 by CRISPR/Cas9 in A549, including phase images (top) and graphic quantifications (bottom). Data are represented as mean ± SEM ( n = 5). (H) Western blotting analysis of EMT and cancer stemness markers by CRISPR/Cas9 in A549 cells. (I) The protein expression levels of TXNRD1, cleaved Caspase 3, Caspase 3, cleaved PARP, and the total form of PARP were determined by Western blotting by CRISPR/Cas9 in A549 cells.

Article Snippet: Primary antibodies against TXNRD1, EMT, and cancer stem cell (CSC)-related genes (diluted 1:1000) were purchased from ABclonal: TXNRD1 (A4725) and Cas9 (A23005).

Techniques: CRISPR, FACS, Flow Cytometry, Transfection, Single Cell, Western Blot, Expressing, Control, Knockdown, Migration, Microscopy, Tube Formation Assay

Journal: Cancer Biology & Therapy

Article Title: Thioredoxin reductase-1 facilitates cellular plasticity and stemness mediated by TGF-β1 in non-small cell lung cancer

doi: 10.1080/15384047.2026.2724630

Figure Lengend Snippet: TXNRD1 modulates TGF-β1 signaling and autocrine function in NSCLC cells. (A) Growth factor analysis of TXNRD1-WT and TXNRD1-sg2 groups in A549 cells. Forty-one growth factors were detected, and the significantly changed factors were graphed (red box denotes the TGF-β1 factors). (B) Secreted cytokine TGF-β1 was measured by ELISA assays in the concentrated conditioned medium of H1975 and H1299 cells transfected with TXNRD1 expression constructs and CRISPR/Cas9 knockdown of TXNRD1 in A549 cells. Data represent the mean ± SEM ( n = 5). All data statistics were based on * p < 0.05, ** p < 0.01, and *** p < 0.001 calculated by one-way ANOVA with the Brown–Forsythe test. (C) A heatmap plot of the relative expression profiles of the TGF-β1 downstream signaling pathway in H1975 and A549 cells was detected by qRT-PCR. (D) A heatmap plot of the relative expression profiles of the TXNRD1 and EMT marker signaling in TGF-β1-induced A549 cells (0, 5, or 10 ng/ml for 48 h) was detected by qRT-PCR. (E) Ingenuity pathway analysis (IPA) of the top upstream regulators. Bar chart depicting the top predicted upstream regulators and signaling pathways based on RNA-seq data. The y-axis lists the primary regulatory networks identified (including CREB1/E2F/MYC, TGF-β/YAP1, FOXM1, ERBB2, and TP53/TP63). Red bars extending to the right (positive values) denote predicted pathway activation, while blue bars extending to the left (negative values) denote predicted pathway inhibition. (F) Results of GSE After RNA-sequencing analysis for hallmark analysis, enriched gene sets were shown after transcriptome analysis with the TGF-β1 and SMAD genes signaling origin from TXNRD1-WT and the knockdown of sgTXNRD1 in A549 cells. (G) Heatmap of gene expression based on the intensity of TXNRD1-WT and sgTXNRD1 with/without TGF-β1 induction in A549 cells. The genes are listed on the y-axis, and the conditions are shown on the x-axis. The color scale at the bottom provides a guide to interpret the expression levels, with the range of colors from blue (low) to red (high). This type of plot is commonly used to identify patterns of gene expression, such as genes that are consistently up-regulated or down-regulated in matched gene sets, respectively. (H) Western blotting analysis of the indicated EMT and cancer stemness markers by CRISPR/Cas9 with/without 10 ng/ml TGF-β1 for 48 h in A549 cells.

Article Snippet: Primary antibodies against TXNRD1, EMT, and cancer stem cell (CSC)-related genes (diluted 1:1000) were purchased from ABclonal: TXNRD1 (A4725) and Cas9 (A23005).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Construct, CRISPR, Knockdown, Quantitative RT-PCR, Marker, Protein-Protein interactions, RNA Sequencing, Activation Assay, Inhibition, Gene Expression, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Thioredoxin reductase-1 facilitates cellular plasticity and stemness mediated by TGF-β1 in non-small cell lung cancer

doi: 10.1080/15384047.2026.2724630

Figure Lengend Snippet: Loss of TXNRD1 impaired metastatic tumor growth in an orthotopic model of lung cancer. (A) Representative bioluminescence imaging of lung colonization in mice injected with TXNRD1-WT cells, TXNRD1-sg cells, and TXNRD1-sg cells treated with a TRi-1 inhibitor at 6 and 12 weeks after transplantation. The color scale indicates the photon flux intensity from low (purple) to high (red). (B) Quantitative analysis of the bioluminescence signal intensity over time (mean ± SEM, n = 5/group). (C) Tumor nodules were measured at the experimental endpoint after cell transplantation. (D) Kaplan–Meier survival curves comparing the TXNRD1-WT (black), TXNRD1-deficiency group (blue), and TXNRD1-deficiency plus TRi-1 treatment (red) groups in an orthotopic model of lung cancer ( n = 5/groups). Statistical significance was determined by Student’s t-test or log-rank test; p < 0.05 was considered significant. (E) A schematic representation depicting the role of transforming growth factor-β1 (TGF-β1) signaling in facilitating epithelial–mesenchymal transition (EMT) and the production of cancer stem cell (CSC)-like genes in non-small cell lung cancer (NSCLC). Upon binding to the TGF- β receptor (TGF-βR), TGF-β1 activates thioredoxin reductase 1 (TXNRD1), thereby enhancing the phosphorylation of SMAD2/3 and promoting the transcription of EMT/CSC-related genes, resulting in epithelial cells adopting a mesenchymal phenotype. The CRISPR/Cas9-induced deletion of TXNRD1 inhibits this pathway, disrupting EMT/CSC gene activation and thereby enhancing apoptosis in cancer cells and delaying tumor progression in NSCLC. This figure was generated and exported using Google NotebookLM. The image bears the “NotebookLM” watermark in the bottom-right corner, indicating that it was generated by NotebookLM's built-in AI visualization workflow using the inputs provided within the NotebookLM environment.

Article Snippet: Primary antibodies against TXNRD1, EMT, and cancer stem cell (CSC)-related genes (diluted 1:1000) were purchased from ABclonal: TXNRD1 (A4725) and Cas9 (A23005).

Techniques: Imaging, Injection, Transplantation Assay, Binding Assay, Phospho-proteomics, CRISPR, Activation Assay, Generated

Journal: bioRxiv

Article Title: Lineage recording reveals hijacked hepatic progenitor states as a common origin of HCC and ICC

doi: 10.64898/2026.07.13.738237

Figure Lengend Snippet: (a) Schematic design of the CRISPR-Cas9 Evolving Recording Arrays Multiplexed by Csy4 (CERAMIC) lineage-recording system. A multiplexed array of 24 sgRNAs is driven by a U6P promoter. The core recording construct features a doxycycline-inducible Cas9 cassette (under TetO control), an Ef1a-driven Teton3G transactivator, a Csy4 endoribonuclease, and a ∼3000-bp synthetic target array comprising eight tandem modules (TA1-TA8). Each TA module is engineered with a Csy4 cleavage site, an sgRNA target site, and a poly(A) capture element. Subsequent Csy4 processing cleaves the continuous array transcript into individual ∼300-bp barcode transcripts, enabling co-capture of lineage records alongside endogenous cellular mRNAs via droplet-based single-cell RNA sequencing (scRNA-seq). (b) Frequency distribution of editing outcomes recovered from CERAMIC mouse embryonic stem cells (ESCs) after 48 h of induction with 100 ng/mL Dox, classified by the number of affected target sites and mutation types. The y-axis represents the log2-transformed frequency of each editing event, while the x-axis indicates the size of insertions or deletions (in base pairs, bp). Color-coded bars distinguish single- and multi-target-site deletions (above the baseline) and single-target-site insertions (below the baseline). (c) Representative editing profiles across the target-site modules (TA1-TA8). A consolidated profile of 100 representative edited CERAMIC alleles generated in CERAMIC mouse embryonic stem cells (ESCs) after 48 h of induction with 100 ng/mL doxycycline (Dox), compiled from the experiments shown in . Each row represents an individual edited allele, with insertions, deletions, and unedited regions shown in blue, red, and light gray, respectively. In the schematic profile positioned above the allele matrix, dark gray bars demarcate the precise double-strand break (DSB) windows targeted by the sgRNAs, while the light gray regions define the broader windows permissible for genetic editing. (d) Experimental workflow for lineage-resolved profiling of liver tumor initiation. CERAMIC mice are subjected to hydrodynamic tail-vein injection of three distinct transposon plasmids: the SB100 plasmid (blue vector) expressing the SB100 transposase protein, the N-RasV12 oncogene co-expressed with a red fluorescent protein on a single vector (red vector), and the myr-AKT oncogene co-expressed with a green fluorescent protein on a single vector (green vector). Black segments within the vectors represent the oncogene sequences. Liver tissues were collected at Days 4, 9, 14, and 19 after hydrodynamic tail-vein injection. (e) Integrated multi-omics strategy to track tumor evolution spanning the hepatic lobule architecture from the central vein (CV) to the portal node (PN). Following hydrodynamic tail-vein injection of the oncogenes into CERAMIC mice, a subset of hepatocytes along the CV-PN axis integrates the oncogenes (represented as purple hepatocytes), while other hepatocytes remain unintegrated (represented as pink hepatocytes). Upon subsequent doxycycline induction, Cas9 expression is activated within the cells, forming a complex with the sgRNAs to cleave the target arrays and generate heritable genetic scars. Double-positive oncogene-expressing hepatocytes are sequentially harvested at successive experimental time points for single-cell RNA sequencing, while parallel liver tissue sections are processed for spatial transcriptomics. Single-cell RNA sequencing and spatial transcriptomics were performed at the indicated time points, and the resulting datasets were used for downstream lineage and spatial analyses. (f) UMAP embedding of the integrated single-cell cellular atlas, resolving major hepatic, stromal, and immune populations within the tumor microenvironment, including hepatocytes, cholangiocytes, liver vascular endothelial cells (LVECs), fibroblasts, hepatic stellate cells (HSCs), Kupffer cells (KCs), lipid-associated macrophages (LAMs), monocytes, neutrophils, T cells, and B cells. (g) Representative reconstructed lineage phylogeny and spatial clonal architecture at Day 4 post-induction integrated with functional single-cell parameters. The circular phylogenetic tree is organized into multi-layered concentric rings mapped to individual cells: the innermost layer displays a rainbow of distinct colors representing individual clones; the second layer indicates the inferred clonal growth advantage (Fitness score); the third layer reflects the single-cell developmental potential (scPlasticity); the fourth layer shows the CytoTRACE2 differentiation score; and the outermost layer specifies the corresponding cell types across the tree.

Article Snippet: A plasmid mixture containing 10 μg pT3-EF1α-myr-Akt, 10 μg pT-CAGGS-NRAS, and 5 μg SB100X transposase was prepared in 2.2 mL sterile 0.9% NaCl and rapidly injected into the lateral tail vein within approximately 10 s. To ensure efficient Cas9 activation before lineage recording, doxycycline (ABclonal, Cat# D48978) was administered one week prior to HTVI by intraperitoneal injection (2 mg per 20 g body weight) every two days.

Techniques: CRISPR, Construct, Control, Single Cell, RNA Sequencing, Mutagenesis, Transformation Assay, Generated, Injection, Plasmid Preparation, Expressing, Biomarker Discovery, Spatial Transcriptomics, Functional Assay, Clone Assay

Journal: bioRxiv

Article Title: Lineage recording reveals hijacked hepatic progenitor states as a common origin of HCC and ICC

doi: 10.64898/2026.07.13.738237

Figure Lengend Snippet: (a) Editing efficiency across the eight individual target array modules (TA1-TA8) responsive to doxycycline induction. The curves plot the editing frequencies calculated from CERAMIC mouse embryonic stem cells (ESCs) following 48 h of induction across a gradient of doxycycline concentrations. The y-axis represents the percentage of edited alleles (%), and the x-axis denotes the doxycycline doses (0, 10, 100, and 1000 ng/mL). Distinct color-coded lines represent the editing efficiencies of the eight target-array (TA) modules across the indicated doxycycline concentrations. (b) UpSet plot illustrating the intersection and overlap of unique edited barcode species across five independent experimental replicates (exp1-exp5) in CERAMIC mouse embryonic stem cells (ESCs) following 96 h of induction with 100 ng/mL doxycycline. The horizontal bars on the bottom-left denote the total barcode pool size captured per experiment; the vertical bars and upper labels indicate the exact intersection size for each overlapping matrix combination. (c) Absolute numbers of unique edited barcodes across target positions t1-t24. Barcodes containing deletions larger than 400 nt were excluded. The plot represents a comprehensive compilation of all edited patterns pooled from the ESC characterization assays and the in vivo single-cell and spatial transcriptomics datasets . Barcodes are color-coded according to five structural categories: intersite deletion (orange; >150 nt), intersite indel (purple; >150 nt deletion with concurrent insertions), intrasite deletion (blue; ≤150 nt), intrasite indel (green; concurrent insertions and ≤150 nt deletions), and insertion (yellow; insertion-only). (d) Relative percentage composition of the four unique edited barcode categories across target positions t1-t24, utilizing the identical aggregated multi-experiment dataset as in (c). The stacked bar chart shows the relative proportions of the four barcode categories across target positions t1-t24. (e) Experimental workflow of the serial-passaging assay using CERAMIC mouse embryonic stem cells (ESCs). The schematic on the left illustrates the multi-promoter array driving 24 sgRNAs paired with an EF1a-driven target array (TS), where continuous Cas9-mediated editing introduces genetic scars into the target array. At Generation 0 (Day 0), Cas9 expression is induced. Cells are sequentially passaged from P1 through P9 at defined intervals (D0.5, D1, D2, D3, D4, D6, D8, D10, and D12). (f) Cumulative numbers and composition of unique pure edit patterns across serial passages (P1-P9). The upper black line and corresponding values indicate the cumulative number of unique pure edit patterns detected from P1 up to each indicated passage, reaching a total of 26,530 unique indels by P9. The area under the curve breaks down the composition at each specific passage: orange represents newly observed pure edit patterns (New Indel) first detected at that time point, while blue represents inherited pure edit patterns (Inherited Indel) shared with prior passages. (g) Matrix of pie charts resolving the proportion of newly emerged versus inherited unique pure edit patterns across separate passages and target sites. Rows represent passages P1-P9, and columns indicate individual sgRNA target sites sg1-sg24. Each pie chart details the specific composition of unique pure edit patterns (defined solely by their indel structural configuration without site prefixes) at that coordinate. Orange wedges indicate newly observed edit patterns, whereas blue wedges indicate edit patterns detected in previous passages. (h) Detection and editing status of the CERAMIC recorder during cellular reprogramming of E13.5 mouse embryonic fibroblasts (MEFs). The stacked bar chart on the left illustrates the percentage of cells with detected barcodes across three independent single-cell RNA-seq replicates (Rep1-Rep3), classified into cells with edited barcodes (dark blue) and those with unedited detected barcodes only (light blue). The pie chart on the right displays the overall composition pooled across all replicates within the across all transcriptome-qualified cells, distinguishing between cells containing edited barcodes, unedited detected barcodes only, and those with transcriptome only. (i) Integrity assessment of target array capture across the three single-cell reprogramming replicates. The stacked bar chart on the left quantifies the proportion of cells recovering different numbers of target array (TA) modules (ranging from 1 to 8, color-coded by a pink-to-dark-purple gradient) within each replicate (Rep1-Rep3). The pie chart summarizes the distribution of recovered TA modules across all three replicates. (j) Experimental schematic and western blot analysis showing the tissue-specific expression of Cas9 protein across multiple organs. CERAMIC mice were subjected to doxycycline (Dox) treatment delivered via drinking water at a concentration of 2 mg/mL for 2 weeks to induce systemic recording (top schematic). Representative western blot images (bottom) display Cas9 protein expression (∼150 kDa) across the various harvested tissues indicated above, with GAPDH (∼35 kDa) serving as the internal loading control and PC representing the positive control lane. (k) Quantification of allele editing efficiencies across diverse organs in the presence or absence of doxycycline. The scatter dot plot illustrates the percentage of edited alleles (%) across the indicated tissues. Light gray circles represent mice without doxycycline treatment (No Dox), and dark gray circles represent doxycycline-treated mice (Dox). Data points represent individual biological replicates, with error bars indicating mean ± s.e.m.

Article Snippet: A plasmid mixture containing 10 μg pT3-EF1α-myr-Akt, 10 μg pT-CAGGS-NRAS, and 5 μg SB100X transposase was prepared in 2.2 mL sterile 0.9% NaCl and rapidly injected into the lateral tail vein within approximately 10 s. To ensure efficient Cas9 activation before lineage recording, doxycycline (ABclonal, Cat# D48978) was administered one week prior to HTVI by intraperitoneal injection (2 mg per 20 g body weight) every two days.

Techniques: In Vivo, Single Cell, Spatial Transcriptomics, Passaging, Expressing, RNA Sequencing, Western Blot, Concentration Assay, Control, Positive Control