human embryonic kidney cell line hek293t  (ATCC)

Journal: Nucleic Acids Research

Article Title: Spatially concentrated adenine base editors efficiently correct PLP1 mutations in oligodendrocytes

doi: 10.1093/nar/gkag156

Figure Lengend Snippet: Development and optimization of concentrated adenine base editors (cABEs). ( A ) Schematic of nSp-ABE8e, eNme2-C ABE8e, and SunTag-integrated cABE variants. nSp-ABE8e was modularized into TadA*-nSpCas9 and the sgRNA module to generate the double plasmids system (DPS) nSp-ABE8e. cABE variants were engineered by fusing GCN4 peptides to the N- or C- terminus of nSp-Cas9 or eNme2-C Cas9, with scFV fused to TadA* to enable multivalent SunTag-mediated recruitment. Detailed structures of each cABEs as shown in ( A ). ( B ) Total fold change in editing activity of nSp-cABE variants relative to nSp-ABE8e (ABE8e) ( n = 3 independent biological replicates). ( C ) Editing windows of ABE8e, N1-nSp-ABE8e, DPS, and cABE-1.0 ( n = 3 independent biological replicates). ( D ) Total fold changes in editing activity of eNme2-C based cABE variants compared to eNme2-C ABE8e ( n = 3 independent biological replicates). ( E ) Editing windows of eNme2-C ABE8e, N1-eNme2-C-ABE8e and cABE-2.0 ( n = 3 independent biological replicates). ( F ) Editing efficiencies of cABE-1.0 and cABE-2.0 at 10 PAM-matched N4CN/NGG sites in HEK293T cells. The leftmost column represents aggregated data from all 10 sites, with subsequent columns grouped by specific PAMs ( n = 3 independent biological replicates). ( G ) Editing windows of cABE-1.0 and cABE-2.0 ( n = 3 independent biological replicates). Data are presented as mean ± SD in panels ( B-G ). P -values were calculated using one-way ANOVA ( B ) or two-tailed Student’s t -test ( D, F ). * P <.05, ** P <.01, *** P <.001.

Article Snippet: Total RNA was isolated from human HEK293T cell lines or mouse NPC-derived OPCs using TRNzol Universal reagent (TIANGEN, DP424) following the manufacturer’s protocol.

Techniques: Activity Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Spatially concentrated adenine base editors efficiently correct PLP1 mutations in oligodendrocytes

doi: 10.1093/nar/gkag156

Figure Lengend Snippet: Spatial concentration of cABEs reduced cytoplasmic deaminase distribution by forming LLPS-like nuclear puncta. ( A ) Schematic illustrating subcellular localization patterns of ABE variants the dynamic behavior of puncta in the presence or absence of 1,6-HD. ( B ) Representative images of ABE 8e and cABE-2.0 showing eGFP-fused scFv-TadA* (green), HA-tagged Cas9 (red), and DAPI (blue) in HEK293T cells. Scale bar, 10 μm. ( C ) Line-scan analyses of relative fluorescence intensity demonstrating spatial co-localization of HA-tagged Cas9 and TadA*-GFP within nuclear puncta formed by cABE-2.0. ( D ) Representative images showing TadA*-GFP puncta formed by cABE-2.0 before and after treatment with 7.5% 1,6-HD. A representative punctum is indicated by a white box. Scale bars, 10 μm. ( E ) Quantification of fluorescence recovery following 1,6-HD treatment, showing relative GFP intensity over time within the puncta regions in ( D ). 1,6-HD was added or washed at the indicated time points. Mean recovery times were calculated from the fluorescence curves. Data are presented as mean ± SEM. n = 5 independent puncta from five independent cells per group. ( F ) Distribution of puncta number per cell from each ABE variant. Cells were categorized based on puncta number and normalized to the total number of analyzed cells. n = 56 independent cells per group pooled from three independent biological experiments. Data are presented as mean ± SD. ( G ) Comparison of average puncta number per cell (left, green) and corresponding genome editing efficiency at Site18 (right, red) across ABE variants. Data are presented as mean ± SD for each group.

Article Snippet: Total RNA was isolated from human HEK293T cell lines or mouse NPC-derived OPCs using TRNzol Universal reagent (TIANGEN, DP424) following the manufacturer’s protocol.

Techniques: Concentration Assay, Fluorescence, Variant Assay, Comparison

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a, Colony survival of control (sgROSA) and ABRAXAS KO HEK293T cells treated with the indicated doses of CPT alone or in combination with 200 nM ATMi. Relative colony survival (%) in individual genotypes and treatment conditions are shown. Data represent mean ± SD of n =3 independent experiments. b, Line graph showing relative colony survival of control (sgHPRT1), ABRAXAS KO, ATM KO and ATM-ABRAXAS double KO (DKO) HT-29 cells exposed to the indicated doses of CPT. Data represent mean ± SD of n =3 independent experiments. c, Representative images of RPA immunofluorescence in control (sgHPRT1) and ABRAXAS KO cells treated with 50 nM CPT +/- 250 nM ATMi for 1 h. Cells were labeled with EdU (10 µM) to identify S-phase cells. Scale bar is shown. d, Scatter plot showing significantly reduced RPA foci in CPT-treated EdU positive S-phase cells upon ATMi. ABRAXAS KO significantly restored RPA foci under these conditions. Data represent mean ± SEM derived from n ≥ 200 EdU positive nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test. e, Schematic showing experimental scheme of native IdU assay. Cells were treated with 50 nM CPT +/- 250 nM ATMi as indicated. IdU was added 20 minutes after CPT addition to label newly synthesized DNA encountering CPT-induced lesions. Scatter plot showing quantification of native IdU experiment. Nascent ssDNA (IdU foci) was significantly reduced in either ATM inhibited or ATM KO cells. ABRAXAS KO significantly restored ssDNA under these conditions. Data represent mean ± SEM derived from n ≥ 180 cells examined over two independent experiments; p values are indicated, two-tailed Mann–Whitney test. f, Scatter plot showing reduced BRCA1 foci in S404A/S406A ABRAXAS expressing cells as compared to WT. WT and S404A/S406A ABRAXAS expressing HT-29 cells were treated with CPT (1 µM) alone or in combination with ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. Experiments were performed four times with similar results. Data represent mean ± SEM derived from n ≥ 150 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. g, ABRAXAS S404A/S406A mutations restore end resection in ATM-inhibited cells. Scatter plot represents quantification of RPA foci in S404A/S406A cells. Experiments were performed as described in (f) and repeated three times with similar results. Data represent mean ± SEM derived from n ≥ 110 cells examined over two repeats; p values are indicated, unpaired two-tailed t test. h, Clonogenic survival assay showing CPT+ATMi resistance in HT-29 cells expressing S404A/S406A ABRAXAS. Data are mean ± SD from n =3 independent experiments.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Control, Immunofluorescence, Labeling, Derivative Assay, Two Tailed Test, Synthesized, MANN-WHITNEY, Expressing, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a, Western blotting showing diminished CPT-induced phospho (S824)-KAP1 (p-KAP1) levels upon AZD0156 treatment, confirming potency of the ATM inhibitor. Control (sgROSA) and ABRAXAS KO HEK293T cells were treated with CPT (1 µM) +/- AZD0156 (250 nM) for 40 min and analyzed by Western blotting using the indicated antibodies. GAPDH serves as loading control. b, Colony survival assay showing CPT+ATMi resistance upon ABRAXAS KO in HEK293T cells. Experiments were repeated n =3 times with similar results. c, Western blotting validating ATM and ABRAXAS DKO in HT-29 cells. GAPDH serves as loading control. d, Colony survival assay showing CPT resistance in ATM and ABRAXAS DKO HT-29 cells. Experiments were repeated n =3 times with similar results. e, Colony survival assay showing olaparib+ATMi resistance in ABRAXAS KO HEK293T cells. Experiments were repeated n =3 times with similar results.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Western Blot, Control, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a, Clonogenic survival assays showing CPT+ATMi resistance in HT-29 cells expressing ABRAXAS S404A/S406A mutant. b, Western blots showing ABRAXAS levels in ABRAXAS KO HEK293T cells complemented with WT or S404A/S406A ABRAXAS. GAPDH serves as loading control. c, Clonogenic survival assay showing CPT+ATMi resistance in ABRAXAS S404A/S406A expressing HEK293T cells.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Expressing, Mutagenesis, Western Blot, Control, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a, Experimental scheme of the iPOND-MS in control (sgROSA) and ABRAXAS KO cells treated with CPT (1 µM) + ATMi (250 nM). ATMi was added 5 min before CPT treatment and present throughout the experiment. (b and c), iPOND purified samples (5% beads) were analyzed by silver staining (b) and Western blotting (c). Enrichment of replisome component PCNA in iPOND purified samples but not in control no click indicates specificity of the iPOND purification. d, Scatter plot showing iPOND-MS data ( n =5). X axis shows log2 fold change (FC) of proteins in ABRAXAS KO cells over sgROSA. Y axis shows average log2 intensity of proteins across sgROSA and ABRAXAS KO cells. e, Gene-annotation clusters network showing top 10 significantly enriched GO terms for the upregulated proteins in ABRAXAS KO cells. f, Experimental scheme of BrdU-coupled chromatin accessibility assay in HEK293T cells treated with CPT (1 µM) +/- ATMi (250 nM). g, Representative gel images showing increased MNase accessibility at BrdU-labeled damaged forks in ABRAXAS KO HEK293T cells. Equal number (∼30 X10 6 ) of control (sgROSA) and ABRAXAS KO cells were treated with CPT (1 µM) + ATMi (250 nM) and labeled with BrdU. BrdU-labeled nuclei were digested with MNase (250 gel units) for the indicated time followed by chromatin isolation. Equal amount of digested chromatin was run on 1.2% agarose gel (left panel), stained with Ethidium bromide (EtBr) and subsequently transferred to nylon membrane by Southern blotting. MNase accessibility at BrdU labeled damaged chromatin was determined by Western blotting using an anti-BrdU antibody (right panel). h, Bar graph showing increased BrdU-labeled mononucleosome fractions in ABRAXAS KO cells as compared to control. Relative percentage of BrdU-labeled mononucleosome fractions normalized to total amount of BrdU-labeled DNA per lane is shown. Data represent mean ± SEM of n =3 independent experiments; p values are indicated, unpaired two-tailed t test. (i and j), ABRAXAS complementation in ABRAXAS KO cells reduced MNase accessibility at BrdU-labeled damaged chromatin. Nuclei isolated from ABRAXAS KO and ABRAXAS-complemented HEK2923T cells were treated with CPT (1 µM) + ATMi (250 nM) and were subjected to MNase digestion for 10 min followed by Southern-Western blotting ( i ). Bar graph showing decreased BrdU-labeled mononucleosome fractions upon ABRAXAS complementation ( j ). Data represent mean ± SEM of n =3 independent experiments; p values are indicated, unpaired two-tailed t test.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Control, Purification, Silver Staining, Western Blot, Labeling, Isolation, Agarose Gel Electrophoresis, Staining, Membrane, Southern Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a LTM-dependent degradation of viral NS1 protein. Western blot analysis shows reduced NS1 protein levels in cells expressing NS1-N LTM compared with the N LTM mutant , while NS1 mRNA levels remain comparable ( n = 3). b Lysosome dependence of LTM-mediated NS1 degradation. NS1-N LTM protein degradation is blocked by lysosomal inhibitors but not by proteasome or autophagy inhibition. HEK293T cells expressing either NS1-N LTM (left) or NS1-N LTM mutant (right) protein were cultured with or without the proteasome inhibitor MG-132 (10 µM), the autophagy inhibitor 3-methyladenine (3-MA; 10 mM), bafilomycin A1 (Baf A1; 0.4 µM), or chloroquine (CQ; 50 µM) for 6 h. The viral NS1 protein was detected by Western blotting ( n = 3). c Co-immunoprecipitation demonstrating interaction of HSC70 with NS1-N LTM but not with the NS1 LTM mutant ( n = 3). d Dependence of LTM-mediated NS1 degradation on LAMP2A. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing NS1-N LTM (left) or NS1-N LTM mutant (right) protein and collected 24 h post-transfection. Viral NS1 protein was detected by Western blotting ( n = 3). Immunofluorescence analysis showing colocalization of NS1-N LTM with HSC70 ( e ) and LAMP2A ( f ), but not of the NS1-N LTM mutant . Green, NS1; red, HSC70 or LAMP2A; blue, nuclei; yellow, colocalization sites; scale bar, 5 µm. Representative images of at least three independent experiments are shown. LAMP2A-dependent degradation of NS1-N LTM during viral infection in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Replication competence of NS1-N LTM and NS1-N LTM mutant viruses in conventional and LAMP2A-KO HEK293T ( i ) and A549 ( j ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of NS1-N LTM or NS1-N LTM mutant virus in conventional and LAMP2A-KO cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( i ) and ( j ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The MDCK (CRL-2936) and HEK293T (CRL-3216) cell lines were purchased from the American Type Culture Collection; A549 cells (ab255450) were provided by Abcam.

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Cell Culture, Immunoprecipitation, Transfection, Construct, Immunofluorescence, Infection, Staining, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a Schematic illustration depicting the design and generation of LYTAR 2.0 influenza viruses. LYTAR 2.0 viruses are attenuated in conventional cells through LTM-mediated viral protein degradation by the CMA system but can replicate efficiently in LAMP2A-knockout (KO) cells. VP stands for viral protein. b Western blots showing the dependence of LTM-mediated viral protein degradation on the lysosome. HEK293T cells expressing the indicated viral proteins were cultured in the presence or absence of Baf A1 (0.4 µM) for 6 h. The viral proteins were detected by Western blotting ( n = 3). c Western blots showing the LAMP2A dependency of LTM-mediated viral protein degradation. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing the indicated viral proteins and collected 24 h post-transfection for viral protein detection by Western blotting ( n = 3). d Western blots showing the LAMP2A dependency of LTM-mediated viral protein degradation in HEK293T cells. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were infected with the indicated LYTAR 2.0 viruses and collected 48 h post-infection for detection of viral proteins by Western blotting ( n = 3). e Western blots showing the LAMP2A dependence of LTM-mediated viral protein degradation in A549 cells. Conventional A549 cells and LAMP2A-KO A549 cells were infected with the indicated LYTAR 2.0 viruses and collected 48 h post-infection for detection of viral proteins by Western blotting ( n = 3). Source data are provided as a Source Data file.

Article Snippet: The MDCK (CRL-2936) and HEK293T (CRL-3216) cell lines were purchased from the American Type Culture Collection; A549 cells (ab255450) were provided by Abcam.

Techniques: Knock-Out, Western Blot, Expressing, Cell Culture, Transfection, Construct, Infection

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a Multi-cycle replication kinetics of the indicated viruses in conventional and LAMP2A-KO MDCK cells. Data are presented as means ± s.d ( n = 3). b triLTM-dependent degradation of viral proteins. Western blot analysis shows reduced levels of triLTM-tagged viral proteins compared with mutated triLTM-tagged controls, while corresponding mRNA levels remain comparable ( n = 3). c Lysosome dependence of triLTM-mediated viral protein degradation. HEK293T cells expressing triLTM-tagged or mutated triLTM-tagged viral proteins were cultured in the presence or absence of Baf A1 (0.4 µM) for 6 h and collected for detection of indicated viral proteins by Western blotting ( n = 3). d Co-immunoprecipitation demonstrating interaction of HSC70 with triLTM-tagged viral proteins but not with mutated triLTM-tagged viral proteins ( n = 3). LAMP2A-dependent degradation of triLTM-tagged viral proteins during viral infection in conventional and LAMP2A-KO HEK293T ( e ) and A549 ( f ) cells ( n = 3). Conventional cells and LAMP2A-KO cells were infected with LYTAR 2.0 dual triLTMs or LYTAR 2.0 dual triLTMs mutant virus and collected at 48 h after infection for detection of indicated proteins by Western blotting ( n = 3). Replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant viruses in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant virus in conventional and LAMP2A-KO HEK293T cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( g ) and ( h ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The MDCK (CRL-2936) and HEK293T (CRL-3216) cell lines were purchased from the American Type Culture Collection; A549 cells (ab255450) were provided by Abcam.

Techniques: Western Blot, Expressing, Cell Culture, Immunoprecipitation, Infection, Mutagenesis, Virus, Immunofluorescence, Staining, Two Tailed Test