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ATCC bj 5ta
Bj 5ta, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 451 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/bj 5ta/product/ATCC
Average 96 stars, based on 451 article reviews

bj 5ta - by Bioz Stars, 2026-06

96/100 stars

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A-B: Western blot showing levels of MTPAP and mitochondrial complex I (CI) NDUFB8, II (CII) SDHB, III (CIII) CYTB, IV (CIV) COX2, V (CV) ATP5B proteins in lysates of fibroblasts from patient P3, P4 and P5 compared to fibroblasts from 3 healthy donor (controls) ( A ), and in lysates of BJ-5ta deleted for MTPAP by CRISPR/Cas9 using 3 different single guides (sg#1, #2, #3) compared to controls ( B ). Vinculin is used as a loading control. Image representative of 3 different experiments. C: Left, representative confocal images of the mitochondrial network stained with mitochondrial outer membrane protein TOMM40 antibody in patient fibroblasts (P3, P4 and P5) and one healthy donor fibroblast (control). Scale bar: 5 µm. Right, quantification of the mitochondrial footprint based on MiNA ImageJ analysis plugin in patient fibroblasts and the average of 3 healthy donor fibroblasts (control), represented as a superplot (each border free-point represents the measure of a cell, each colour is a different experiment). Mean ± SEM of 3 experiments; ns indicates non significance in a paired t-test performed on the average value for each experiment. D: As in C for MTPAP_KO (sg#2) and control cells. E: Left, representative spinning disk live images of TMRE signal in patient fibroblasts (P3, P4 and P5) compared to one healthy donor fibroblast (control). Scale bar: 5 µm. Right, quantification of TMRE signal in patient fibroblasts relative to the average of 3 healthy donor fibroblasts (control). Superplot, mean ± SEM; n=3 experiments (each colour is a different experiment); each border-free point represents the measure of a cell; * indicates p<0.05 in paired t-test performed on the average value for each experiment. F: As in E for MTPAP_KO (sg#2) and control cells. G-H: ATP production ( G ) and basal respiration ( H ) measured by Seahorse mito stress assay in MTPAP_KO (average of sg#1, sg#2 and sg#3 data for each experiment) versus control cells. mean ± SEM; n=3 experiments, each colour is a different experiment, * indicates p<0.05 in Mann-Whitney test. I-J: ISG score (median of fold changes of mRNA expression of 6 ISGs, see Methods) ( I ) and IFNB1 mRNA expression ( J ) in MTPAP_KO cells compared to controls measured by qPCR. Mean ± SEM; n=7 experiments; each colour corresponds to KO_MTPAP cells generated using a single guide RNA targeting MTPAP; ** p<0.01 in Kruskall-Wallis with Dunn’s post-hoc test.

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A-B: Western blot showing levels of MTPAP and mitochondrial complex I (CI) NDUFB8, II (CII) SDHB, III (CIII) CYTB, IV (CIV) COX2, V (CV) ATP5B proteins in lysates of fibroblasts from patient P3, P4 and P5 compared to fibroblasts from 3 healthy donor (controls) ( A ), and in lysates of BJ-5ta deleted for MTPAP by CRISPR/Cas9 using 3 different single guides (sg#1, #2, #3) compared to controls ( B ). Vinculin is used as a loading control. Image representative of 3 different experiments. C: Left, representative confocal images of the mitochondrial network stained with mitochondrial outer membrane protein TOMM40 antibody in patient fibroblasts (P3, P4 and P5) and one healthy donor fibroblast (control). Scale bar: 5 µm. Right, quantification of the mitochondrial footprint based on MiNA ImageJ analysis plugin in patient fibroblasts and the average of 3 healthy donor fibroblasts (control), represented as a superplot (each border free-point represents the measure of a cell, each colour is a different experiment). Mean ± SEM of 3 experiments; ns indicates non significance in a paired t-test performed on the average value for each experiment. D: As in C for MTPAP_KO (sg#2) and control cells. E: Left, representative spinning disk live images of TMRE signal in patient fibroblasts (P3, P4 and P5) compared to one healthy donor fibroblast (control). Scale bar: 5 µm. Right, quantification of TMRE signal in patient fibroblasts relative to the average of 3 healthy donor fibroblasts (control). Superplot, mean ± SEM; n=3 experiments (each colour is a different experiment); each border-free point represents the measure of a cell; * indicates p<0.05 in paired t-test performed on the average value for each experiment. F: As in E for MTPAP_KO (sg#2) and control cells. G-H: ATP production ( G ) and basal respiration ( H ) measured by Seahorse mito stress assay in MTPAP_KO (average of sg#1, sg#2 and sg#3 data for each experiment) versus control cells. mean ± SEM; n=3 experiments, each colour is a different experiment, * indicates p<0.05 in Mann-Whitney test. I-J: ISG score (median of fold changes of mRNA expression of 6 ISGs, see Methods) ( I ) and IFNB1 mRNA expression ( J ) in MTPAP_KO cells compared to controls measured by qPCR. Mean ± SEM; n=7 experiments; each colour corresponds to KO_MTPAP cells generated using a single guide RNA targeting MTPAP; ** p<0.01 in Kruskall-Wallis with Dunn’s post-hoc test.

Article Snippet: Primary fibroblasts (from three healthy donors (controls), and patients 2, 3, 4 and 5) and BJ-5ta fibroblasts (catalog no. CRL-4001; ATCC) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum.

Techniques: Western Blot, CRISPR, Control, Staining, Membrane, MANN-WHITNEY, Expressing, Generated

A-B: ISG score ( A ) and IFNB1 mRNA ( B ) in control and MTPAP_KO (sg#2) cells treated for 10 days with ddC. Mean ± SEM; n=4, each colour is a different experiment; * indicates p<0.05, ** p< 0.01, 2-way ANOVA with Holm-Sidak multiple comparison test. C-D: ISG score ( C ) and IFNB1 mRNA levels ( D ) in BJ-5ta cells double KO IRF3/MTPAP compared to controls. E-F : ISG score ( E ) and IFNB1 mRNA levels ( F ) in MAVS/MTPAP double KO cells compared to controls. G-H : ISG score ( G ) and IFNB1 mRNA levels ( H ) in MDA5/MTPAP double KO cells compared to controls. I-J : ISG score ( I ) and IFNB1 mRNA levels ( J ) in RIG-I/MTPAP double KO cells compared to controls. C-J: Mean ± SEM; n≥4, each colour is a different experiment, KO_MTPAP data are represented by the average of sg#1, sg#2 and sg#3 data; ns indicates non significance, * indicates p<0.05 in Wilcoxon test.

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A-B: ISG score ( A ) and IFNB1 mRNA ( B ) in control and MTPAP_KO (sg#2) cells treated for 10 days with ddC. Mean ± SEM; n=4, each colour is a different experiment; * indicates p<0.05, ** p< 0.01, 2-way ANOVA with Holm-Sidak multiple comparison test. C-D: ISG score ( C ) and IFNB1 mRNA levels ( D ) in BJ-5ta cells double KO IRF3/MTPAP compared to controls. E-F : ISG score ( E ) and IFNB1 mRNA levels ( F ) in MAVS/MTPAP double KO cells compared to controls. G-H : ISG score ( G ) and IFNB1 mRNA levels ( H ) in MDA5/MTPAP double KO cells compared to controls. I-J : ISG score ( I ) and IFNB1 mRNA levels ( J ) in RIG-I/MTPAP double KO cells compared to controls. C-J: Mean ± SEM; n≥4, each colour is a different experiment, KO_MTPAP data are represented by the average of sg#1, sg#2 and sg#3 data; ns indicates non significance, * indicates p<0.05 in Wilcoxon test.

Techniques: Control, Comparison

A: (Left) Relative levels of mtDNA expressed as the averaged relative levels of MT-CO2 , MT-ND1 and mtDNA D-loop signals and ( Right ) of mtRNA expressed as the averaged relative expression levels of MT-ATP6/8 , MT-ND4 , MT-CO1 , MT-CYTB and MT-ND6 in lysates of control cells and of MTPAP_KO cells (sg#2) treated for 10 days with ddC. Mean ± SEM; n=3 experiments, each colour is a different experiment, ** indicates p<0.01, in two-way ANOVA with Holm-Sidak multiple comparison test. B-C-D-J-L-N: Western blot analysis showing protein levels of MTPAP and IRF3 ( B ), STING ( C ), cGAS ( D ), MAVS ( J ), MDA5 ( L ), RIG-I ( N ), in lysates of double KO cells (sg#2 for MTPAP_KO) compared to controls. Vinculin or cofilin are used as loading controls. Pictures representative of 3 different experiments. E: mRNA levels of IFNB1 measured by qPCR in cGAS/MTPAP and STING/MTPAP double KO BJ-5ta cells compared to controls 24 h after stimulation with transfected HT-DNA (sg#2 for MTPAP_KO). Mean ± SEM; n=3 experiments, each colour is a different experiment, **** indicates p< 0.0001 in two-way ANOVA with Holm-Sidak multiple comparison test. F-H: ISG score measured by qPCR in BJ-5ta cells double KO for STING/MTPAP ( F ) cGAS/MTPAP ( H ) cells compared to controls (sg#2 for MTPAP_KO). Mean ± SEM; n≥4 experiments, each colour is a different experiment, MTPAP_KO using sg#2; ns indicates non significance for Wilcoxon tests. G-I: IFNB1 mRNA expression measured by qPCR in double KO STING/MTPAP ( G ) cGAS/MTPAP ( I ) cells compared to controls (sg#2 for MTPAP_KO). N=4 experiments, each colour is a different experiment; ns indicates non significance for Wilcoxon tests. K-M: IFNB1 mRNA expression levels in MAVS_KO ( K ) and MDA5_KO ( M ) cells compared to BJ-5ta controls when transfected with poly(I:C). Mean ± SEM; n=3 experiments, ** indicates p<0.01, *** p< 0.001 in two-way ANOVA with Holm-Sidak multiple comparison test. O: IFNB1 mRNA expression levels in RIG-I_KO BJ-5ta cells compared to controls Infected with Sendai Virus. Mean ± SEM; n=5, ** indicates p<0.01 in two-way ANOVA with Holm-Sidak multiple comparison test.

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A: (Left) Relative levels of mtDNA expressed as the averaged relative levels of MT-CO2 , MT-ND1 and mtDNA D-loop signals and ( Right ) of mtRNA expressed as the averaged relative expression levels of MT-ATP6/8 , MT-ND4 , MT-CO1 , MT-CYTB and MT-ND6 in lysates of control cells and of MTPAP_KO cells (sg#2) treated for 10 days with ddC. Mean ± SEM; n=3 experiments, each colour is a different experiment, ** indicates p<0.01, in two-way ANOVA with Holm-Sidak multiple comparison test. B-C-D-J-L-N: Western blot analysis showing protein levels of MTPAP and IRF3 ( B ), STING ( C ), cGAS ( D ), MAVS ( J ), MDA5 ( L ), RIG-I ( N ), in lysates of double KO cells (sg#2 for MTPAP_KO) compared to controls. Vinculin or cofilin are used as loading controls. Pictures representative of 3 different experiments. E: mRNA levels of IFNB1 measured by qPCR in cGAS/MTPAP and STING/MTPAP double KO BJ-5ta cells compared to controls 24 h after stimulation with transfected HT-DNA (sg#2 for MTPAP_KO). Mean ± SEM; n=3 experiments, each colour is a different experiment, **** indicates p< 0.0001 in two-way ANOVA with Holm-Sidak multiple comparison test. F-H: ISG score measured by qPCR in BJ-5ta cells double KO for STING/MTPAP ( F ) cGAS/MTPAP ( H ) cells compared to controls (sg#2 for MTPAP_KO). Mean ± SEM; n≥4 experiments, each colour is a different experiment, MTPAP_KO using sg#2; ns indicates non significance for Wilcoxon tests. G-I: IFNB1 mRNA expression measured by qPCR in double KO STING/MTPAP ( G ) cGAS/MTPAP ( I ) cells compared to controls (sg#2 for MTPAP_KO). N=4 experiments, each colour is a different experiment; ns indicates non significance for Wilcoxon tests. K-M: IFNB1 mRNA expression levels in MAVS_KO ( K ) and MDA5_KO ( M ) cells compared to BJ-5ta controls when transfected with poly(I:C). Mean ± SEM; n=3 experiments, ** indicates p<0.01, *** p< 0.001 in two-way ANOVA with Holm-Sidak multiple comparison test. O: IFNB1 mRNA expression levels in RIG-I_KO BJ-5ta cells compared to controls Infected with Sendai Virus. Mean ± SEM; n=5, ** indicates p<0.01 in two-way ANOVA with Holm-Sidak multiple comparison test.

Techniques: Expressing, Control, Comparison, Western Blot, Transfection, Infection, Virus

$A: Representative confocal microscopy image of immunostaining with antibody to mitochondrial protein TOMM40 and J2 antibody to dsRNA in control and MTPAP_KO (sg#1) cells. Scale bar: 5 µm. B: Quantification of average pixel intensity of J2 dsRNA signal in mitochondria in control vs MTPAP_KO cells (average of sg#1, #2 and #3). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; * indicates p<0.05 in paired t-test performed on the mean value for each experiment. C: Percentage of dsRNA dots in mitochondria with an area over 1 µm 2 in control cells vs MTPAP_KO cells (average of sg#1, #2 and #3). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; * indicates p<0.05 in paired t-test performed on the mean value for each experiment. D: Representative confocal microscopy image of immunostaining with antibody to mitochondrial RNA granule protein GRSF1 and dsRNA (J2) in control and MTPAP_KO (sg#2) BJ-5ta cells. Scale bar: 5 µm. E: Representative image of the 3D volume reconstruction of STED images after immunostaining with antibody against mitochondrial protein TOMM40 and dsRNA (J2) in control and MTPAP_KO cells (sg#2). Yellow dots correspond to dsRNA particles detected outside of the mitochondrial network. Scale bar: 3 µm. F: Percentage of dsRNA dots detected outside of the mitochondrial network in control vs MTPAP_KO cells (sg#2). Mean ± SEM; n=3 experiments; each point represents the measure of a cell; each colour is a different experiment; **** indicates p<0.0001 in Mann-Whitney test. G: Quantification of the median volume of dsRNA particles detected inside or outside of the mitochondrial network in MTPAP_KO cells (sg#2). Mean ± SEM; n=3 experiments; each point represents the measure of a cell; each colour is a different experiment; *** indicates p<0.001 in Mann-Whitney test. H: Quantification of mtRNA levels measured by qPCR in cytosolic fractions of control and MTPAP_KO cells (sg#2). Mean ± SEM; n=8 experiments; each colour is a different experiment; ns indicates non significance, ** p< 0.01 in 2-way ANOVA with Holm-Sidak multiple comparison test. I-J: ISG score ( I ) and IFNB1 mRNA expression ( J ) measured by qPCR in MTPAP_KO cells (average of sg#1, sg#2 and sg#3 data for each experiment) treated for 24h with 300 µM DIDS compared to controls. Mean ± SEM; n=4 experiments, each colour is a different experiment; * indicates p<0.05, ** p<0.01, two-way ANOVA with Holm-Sidak multiple comparison test.$

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A: Representative confocal microscopy image of immunostaining with antibody to mitochondrial protein TOMM40 and J2 antibody to dsRNA in control and MTPAP_KO (sg#1) cells. Scale bar: 5 µm. B: Quantification of average pixel intensity of J2 dsRNA signal in mitochondria in control vs MTPAP_KO cells (average of sg#1, #2 and #3). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; * indicates p<0.05 in paired t-test performed on the mean value for each experiment. C: Percentage of dsRNA dots in mitochondria with an area over 1 µm 2 in control cells vs MTPAP_KO cells (average of sg#1, #2 and #3). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; * indicates p<0.05 in paired t-test performed on the mean value for each experiment. D: Representative confocal microscopy image of immunostaining with antibody to mitochondrial RNA granule protein GRSF1 and dsRNA (J2) in control and MTPAP_KO (sg#2) BJ-5ta cells. Scale bar: 5 µm. E: Representative image of the 3D volume reconstruction of STED images after immunostaining with antibody against mitochondrial protein TOMM40 and dsRNA (J2) in control and MTPAP_KO cells (sg#2). Yellow dots correspond to dsRNA particles detected outside of the mitochondrial network. Scale bar: 3 µm. F: Percentage of dsRNA dots detected outside of the mitochondrial network in control vs MTPAP_KO cells (sg#2). Mean ± SEM; n=3 experiments; each point represents the measure of a cell; each colour is a different experiment; **** indicates p<0.0001 in Mann-Whitney test. G: Quantification of the median volume of dsRNA particles detected inside or outside of the mitochondrial network in MTPAP_KO cells (sg#2). Mean ± SEM; n=3 experiments; each point represents the measure of a cell; each colour is a different experiment; *** indicates p<0.001 in Mann-Whitney test. H: Quantification of mtRNA levels measured by qPCR in cytosolic fractions of control and MTPAP_KO cells (sg#2). Mean ± SEM; n=8 experiments; each colour is a different experiment; ns indicates non significance, ** p< 0.01 in 2-way ANOVA with Holm-Sidak multiple comparison test. I-J: ISG score ( I ) and IFNB1 mRNA expression ( J ) measured by qPCR in MTPAP_KO cells (average of sg#1, sg#2 and sg#3 data for each experiment) treated for 24h with 300 µM DIDS compared to controls. Mean ± SEM; n=4 experiments, each colour is a different experiment; * indicates p<0.05, ** p<0.01, two-way ANOVA with Holm-Sidak multiple comparison test.

Techniques: Confocal Microscopy, Immunostaining, Control, MANN-WHITNEY, Comparison, Expressing

$A: Representative confocal microscopy image of immunostained mitochondrial protein TOMM40 and dsRNA (J2) in control and in patient P3, P4 and P5 fibroblasts. Scale bar: 5µm. B: Quantification of average pixel intensity of immunostaining of dsRNA signal in mitochondria in control cells and in patients’ cells (P3, P4 and P5). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; ns indicates non significance, * p<0.05 in one-way ANOVA with Dunnett multiple comparison test test performed on the mean value for each experiment C: Percentage of cells with a J2 signal 1.5-fold above the average of 3 controls. Mean ± SEM; n=3 experiments, each colour is a different experiment. D: Representative confocal microscopy image of immunostained TOMM40 and dsRNA (J2) in control and MTPAP_KO BJ-5ta cells (sg#2) untreated (NT) or treated with actinomycin D (ActD) for up to 24h. Scale bar: 5 µm. E: Quantification of pixel intensity of immunostaining of dsRNA signal in mitochondria in control and MTPAP_KO cells (average of sg#1, #2, #3) untreated (NT) and treated with actinomycin D overtime. Mean ± SEM; n=6 experiments, **** indicates p<0.0001 in two-way ANOVA with Holm-Sidak multiple comparison test. F: Representative confocal microscopy image of immunostained TOMM40 and dsRNA (J2) in MTPAP_KO cells (sg#2) non treated (NT), treated in culture for 10 days with ddC, or untreated in culture but treated after fixation with dsRNA specific RNase III (see Methods). Scale bar: 5 µm. G: Representative confocal microscopy image of immunostained PNPT1 and dsRNA (J2) in control and MTPAP_KO cells (sg#3). Scale bar: 5µm. H-I: Quantification of the median sphericity ( H ) and median volume ( I ) of dsRNA particles in control and MTPAP_KO cells (sg#2). Mean ± SEM; n=3, each dot represents a different cell, each colour represents a different experiment, *** indicates p<0.001, **** p<0.0001 in Mann-Whitney test. J: Schematic representation of the experimental set-up to isolate the cytosolic fraction using digitonin and differential centrifugation. K: Western blot analysis of the different fractions generated to isolate cytosolic fractions. Vinculin is used as a cytosolic marker, Lamin A/C as a nuclear fraction marker, TIM44 and TOM40 as mitochondrial membrane markers and TFAM as mitochondrial matrix marker. Note the absence of all markers but vinculin in the cytosolic fraction.$

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A: Representative confocal microscopy image of immunostained mitochondrial protein TOMM40 and dsRNA (J2) in control and in patient P3, P4 and P5 fibroblasts. Scale bar: 5µm. B: Quantification of average pixel intensity of immunostaining of dsRNA signal in mitochondria in control cells and in patients’ cells (P3, P4 and P5). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; ns indicates non significance, * p<0.05 in one-way ANOVA with Dunnett multiple comparison test test performed on the mean value for each experiment C: Percentage of cells with a J2 signal 1.5-fold above the average of 3 controls. Mean ± SEM; n=3 experiments, each colour is a different experiment. D: Representative confocal microscopy image of immunostained TOMM40 and dsRNA (J2) in control and MTPAP_KO BJ-5ta cells (sg#2) untreated (NT) or treated with actinomycin D (ActD) for up to 24h. Scale bar: 5 µm. E: Quantification of pixel intensity of immunostaining of dsRNA signal in mitochondria in control and MTPAP_KO cells (average of sg#1, #2, #3) untreated (NT) and treated with actinomycin D overtime. Mean ± SEM; n=6 experiments, **** indicates p<0.0001 in two-way ANOVA with Holm-Sidak multiple comparison test. F: Representative confocal microscopy image of immunostained TOMM40 and dsRNA (J2) in MTPAP_KO cells (sg#2) non treated (NT), treated in culture for 10 days with ddC, or untreated in culture but treated after fixation with dsRNA specific RNase III (see Methods). Scale bar: 5 µm. G: Representative confocal microscopy image of immunostained PNPT1 and dsRNA (J2) in control and MTPAP_KO cells (sg#3). Scale bar: 5µm. H-I: Quantification of the median sphericity ( H ) and median volume ( I ) of dsRNA particles in control and MTPAP_KO cells (sg#2). Mean ± SEM; n=3, each dot represents a different cell, each colour represents a different experiment, *** indicates p<0.001, **** p<0.0001 in Mann-Whitney test. J: Schematic representation of the experimental set-up to isolate the cytosolic fraction using digitonin and differential centrifugation. K: Western blot analysis of the different fractions generated to isolate cytosolic fractions. Vinculin is used as a cytosolic marker, Lamin A/C as a nuclear fraction marker, TIM44 and TOM40 as mitochondrial membrane markers and TFAM as mitochondrial matrix marker. Note the absence of all markers but vinculin in the cytosolic fraction.

Techniques: Confocal Microscopy, Control, Immunostaining, Comparison, MANN-WHITNEY, Centrifugation, Western Blot, Generated, Marker, Membrane

(A) Schematic of the single-vector V-SWITCH architecture. The CAM (EF1α promoter) encodes NLS–mNG3A(1–10)–PCS–ER anchor–T2A–blasticidin resistance. The NCM (CMV promoter) encodes NLS–mNG(11)–BFP–NLS. Both modules are delivered on a single lentiviral backbone. (B) Principle of single-vector reporter operation, as described for . In this design, the NCM is an exogenous NLS–mNG(11)–BFP fusion expressed from the CMV promoter. (C) Representative images of A549 DENV-SWITCH cells infected with DENV at increasing MOIs (uninfected, 0.3, 1, 5) and co-stained for NS3 (magenta). Reporter fluorescence (V-SWITCH) is shown in green. Scale bar, 20 µm. (D) Percentage of infected cells detected by anti-NS3 immunostaining (black bars) versus reporter fluorescence (white bars) across MOIs. Data are mean ± s.d. (n = 3 independent experiments). (E) Representative flow cytometry plots for DENV-SWITCH A549 cells infected at MOI 5. Left: reporter fluorescence (FITC-A); middle: NS3 immunostaining (PE-A); right: NS3 staining versus reporter fluorescence. BFP-positive cells were gated to define the reporter-expressing population. (F) Dose–response curve for MK0608 in DENV-SWITCH A549 cells. IC₅₀ is indicated. (G) Dose–response curve for niclosamide in DENV-SWITCH A549 cells. IC₅₀ is indicated. Data in (F–G) are mean ± s.d. (n = 3 independent experiments). (H) Effect of siRNA-mediated knockdown of EMC6 and RACK1 on DENV infection in A549 DENV-SWITCH cells at 24 hpi (MOI 5). DENV-infected cells are expressed as a percentage of siCTRL. ***P < 0.001, unpaired two-tailed t-test. Data are mean ± s.d. (n = 3 independent experiments). (I) Percentage of infected cells detected by anti-NS3 immunostaining (black bars) and reporter fluorescence (white bars) in BJ-5α fibroblasts infected with DENV at MOI 1, 5, and 10. Data are mean ± s.d. (J) Effect of HA15 (50 µM), a BiP/GRP78 ATPase domain inhibitor, on DENV infection in BJ-5α DENV-SWITCH cells at 24 hpi. DENV infection (black bars, % relative to DMSO control) and cell viability (white bars) are shown. ns, not significant; ***P < 0.001, unpaired two-tailed t-test. Data are mean ± s.d. (n = 3 independent experiments).

Journal: bioRxiv

Article Title: V-SWITCH: A single-vector OFF-to-ON fluorescent reporter of live RNA virus infections

doi: 10.64898/2026.04.08.717260

Figure Lengend Snippet: (A) Schematic of the single-vector V-SWITCH architecture. The CAM (EF1α promoter) encodes NLS–mNG3A(1–10)–PCS–ER anchor–T2A–blasticidin resistance. The NCM (CMV promoter) encodes NLS–mNG(11)–BFP–NLS. Both modules are delivered on a single lentiviral backbone. (B) Principle of single-vector reporter operation, as described for . In this design, the NCM is an exogenous NLS–mNG(11)–BFP fusion expressed from the CMV promoter. (C) Representative images of A549 DENV-SWITCH cells infected with DENV at increasing MOIs (uninfected, 0.3, 1, 5) and co-stained for NS3 (magenta). Reporter fluorescence (V-SWITCH) is shown in green. Scale bar, 20 µm. (D) Percentage of infected cells detected by anti-NS3 immunostaining (black bars) versus reporter fluorescence (white bars) across MOIs. Data are mean ± s.d. (n = 3 independent experiments). (E) Representative flow cytometry plots for DENV-SWITCH A549 cells infected at MOI 5. Left: reporter fluorescence (FITC-A); middle: NS3 immunostaining (PE-A); right: NS3 staining versus reporter fluorescence. BFP-positive cells were gated to define the reporter-expressing population. (F) Dose–response curve for MK0608 in DENV-SWITCH A549 cells. IC₅₀ is indicated. (G) Dose–response curve for niclosamide in DENV-SWITCH A549 cells. IC₅₀ is indicated. Data in (F–G) are mean ± s.d. (n = 3 independent experiments). (H) Effect of siRNA-mediated knockdown of EMC6 and RACK1 on DENV infection in A549 DENV-SWITCH cells at 24 hpi (MOI 5). DENV-infected cells are expressed as a percentage of siCTRL. ***P < 0.001, unpaired two-tailed t-test. Data are mean ± s.d. (n = 3 independent experiments). (I) Percentage of infected cells detected by anti-NS3 immunostaining (black bars) and reporter fluorescence (white bars) in BJ-5α fibroblasts infected with DENV at MOI 1, 5, and 10. Data are mean ± s.d. (J) Effect of HA15 (50 µM), a BiP/GRP78 ATPase domain inhibitor, on DENV infection in BJ-5α DENV-SWITCH cells at 24 hpi. DENV infection (black bars, % relative to DMSO control) and cell viability (white bars) are shown. ns, not significant; ***P < 0.001, unpaired two-tailed t-test. Data are mean ± s.d. (n = 3 independent experiments).

Article Snippet: A549 (ATCC, CCL-185), HEK 293T (ATCC, CRL-3216), HEK 293FT (Invitrogen, R70007), and BJ-5α (ATCC, CRL-4001) cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% foetal bovine serum (FBS; Gibco) at 37°C in a humidified atmosphere containing 5% CO2.

Techniques: Plasmid Preparation, Infection, Staining, Fluorescence, Immunostaining, Flow Cytometry, Expressing, Knockdown, Two Tailed Test, Control

A) Immunofluorescence for bromouridine (BrU) after cardiomyocytes were cultured with BrU for 2 hr to label nascent RNAs. One binucleated cardiomyocyte per image, with nuclei zoomed in. Texts and icons below images indicate nuclear states. Scale bar: 5 μm. All data in , except , are from cardiomyocytes freshly isolated from mice at day 14 post tamoxifen. B) Nuclear BrU intensity by nuclear states after 1-hr BrU incubation. Density and box plots: BrU intensity distribution and interquartile range. Circles: mean intensity within biological replicates (color coded). Asterisks: P < 0.05 from t -tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 244 intact nuclei from 4 WT mice, 275 intact, 111 ruptured, 104 resealed nuclei from 3 Lmna CKO mice. C) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (519 nuclei from 3 mice). Line: simple linear regression fit with 95% confidence interval. R: Pearson correlation coefficient. P: t -test p -value on linear regression-estimated means with mouse-clustered standard errors. See for individual replicates. D) Immunofluorescence for RNA polymerase II (Pol II) in cardiomyocytes. Scale bar: 5 μm. E) Nuclear Pol II intensity by nuclear states. Underlying data: 285 intact nuclei from 3 WT mice, 180 intact, 133 ruptured, 136 resealed nuclei from 3 Lmna CKO mice. Graph annotations and statistics as in (B) . F) Relationship between Pol II intensity and NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (495 nuclei from 3 mice). Graph annotations and statistics as in (C) . G) Pol II ChIP-seq and input read coverage in WT and Lmna CKO cardiomyocytes (3 mice per genotype). ChIP-seq signals are normalized to internal spike-in controls. H) Average Pol II ChIP-seq signals across 21,177 protein-coding genes. X-axis: 100 equally-spaced bins in gene bodies, 5 bins for 1 kb-upstream regions, and 10 bins for 2 kb-downstream regions. I) Statistical comparison of gene-body Pol II signals in Lmna CKO versus WT cardiomyocytes for 11,942 Pol II-bound genes. Pol II-lost or gained genes are defined at limma p -value < 0.05. J) Ten most enriched Gene Ontology terms among the 1,759 Pol II-lost genes in Lmna CKO cardiomyocytes, with three representative genes for each term. P: Metascape p -value. K) Gene expression state of Pol II-lost, Pol II-gained, and all other genes in Lmna CKO (n=5) versus WT (n=7) hearts. P, DESeq2 p -value. RNA-seq data from En et al. 2024. L) Summary of . Nuclear rupture causes transcriptional deficiency due to RNA Pol II loss.

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Immunofluorescence for bromouridine (BrU) after cardiomyocytes were cultured with BrU for 2 hr to label nascent RNAs. One binucleated cardiomyocyte per image, with nuclei zoomed in. Texts and icons below images indicate nuclear states. Scale bar: 5 μm. All data in , except , are from cardiomyocytes freshly isolated from mice at day 14 post tamoxifen. B) Nuclear BrU intensity by nuclear states after 1-hr BrU incubation. Density and box plots: BrU intensity distribution and interquartile range. Circles: mean intensity within biological replicates (color coded). Asterisks: P < 0.05 from t -tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 244 intact nuclei from 4 WT mice, 275 intact, 111 ruptured, 104 resealed nuclei from 3 Lmna CKO mice. C) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (519 nuclei from 3 mice). Line: simple linear regression fit with 95% confidence interval. R: Pearson correlation coefficient. P: t -test p -value on linear regression-estimated means with mouse-clustered standard errors. See for individual replicates. D) Immunofluorescence for RNA polymerase II (Pol II) in cardiomyocytes. Scale bar: 5 μm. E) Nuclear Pol II intensity by nuclear states. Underlying data: 285 intact nuclei from 3 WT mice, 180 intact, 133 ruptured, 136 resealed nuclei from 3 Lmna CKO mice. Graph annotations and statistics as in (B) . F) Relationship between Pol II intensity and NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (495 nuclei from 3 mice). Graph annotations and statistics as in (C) . G) Pol II ChIP-seq and input read coverage in WT and Lmna CKO cardiomyocytes (3 mice per genotype). ChIP-seq signals are normalized to internal spike-in controls. H) Average Pol II ChIP-seq signals across 21,177 protein-coding genes. X-axis: 100 equally-spaced bins in gene bodies, 5 bins for 1 kb-upstream regions, and 10 bins for 2 kb-downstream regions. I) Statistical comparison of gene-body Pol II signals in Lmna CKO versus WT cardiomyocytes for 11,942 Pol II-bound genes. Pol II-lost or gained genes are defined at limma p -value < 0.05. J) Ten most enriched Gene Ontology terms among the 1,759 Pol II-lost genes in Lmna CKO cardiomyocytes, with three representative genes for each term. P: Metascape p -value. K) Gene expression state of Pol II-lost, Pol II-gained, and all other genes in Lmna CKO (n=5) versus WT (n=7) hearts. P, DESeq2 p -value. RNA-seq data from En et al. 2024. L) Summary of . Nuclear rupture causes transcriptional deficiency due to RNA Pol II loss.

Article Snippet: We used AAVpro 293T cells (TaKaRa, 632273) for MyoAAV production and BJ-5ta cells (human hTERT-immortalized foreskin fibroblast; ATCC, CRL-4001) for ChIP-seq spike-in controls.

Techniques: Immunofluorescence, Cell Culture, Isolation, Incubation, ChIP-sequencing, Comparison, Gene Expression, RNA Sequencing

$A) Number of Pol II ChIP-seq and input sequencing reads aligned to the mouse genome (experimental) or the human genome (spike-in control). Scale factors are computed by spike-in control reads and sequencing depths and used to normalize Pol II ChIP-seq signals. All data in are derived from mice at 2 weeks post tamoxifen. B) Pol II ChIP-seq read coverage in all mouse genes stratified by gene-body Pol II coverage. Genes with Pol II coverage greater than or equal to 100 (2 in Log 10 ) were considered Pol II-bound (11,942 genes). C) Pol II ChIP-seq read coverage in all human genes, derived from the spike-in control chromatin. D) Gene-body Pol II ChIP-seq read coverage between every pair of biological replicates. E) Principal Component Analysis (PCA) of gene-body Pol II coverage in 11,942 Pol II-bound protein-coding genes. F) Cumulative fraction of 1,759 Pol II-lost genes and all other genes (y-axis) along the scale of differential gene expression between Lmna CKO hearts and wild-type hearts (x-axis). P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes. G) Gene expression state of Pol II-lost genes, Pol II-gained genes, and all other genes in the cardiomyocyte population in Lmna CKO (n=3) versus WT (n=3) hearts derived from single-nucleus RNA-seq in En et al. 2024. P, DESeq2 p -value. H) Same as F, but along the scale of differential gene expression between Lmna CKO and wild-type pseudo-bulk cardiomyocytes from the single-nucleus RNA-seq. P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes.$

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Number of Pol II ChIP-seq and input sequencing reads aligned to the mouse genome (experimental) or the human genome (spike-in control). Scale factors are computed by spike-in control reads and sequencing depths and used to normalize Pol II ChIP-seq signals. All data in are derived from mice at 2 weeks post tamoxifen. B) Pol II ChIP-seq read coverage in all mouse genes stratified by gene-body Pol II coverage. Genes with Pol II coverage greater than or equal to 100 (2 in Log 10 ) were considered Pol II-bound (11,942 genes). C) Pol II ChIP-seq read coverage in all human genes, derived from the spike-in control chromatin. D) Gene-body Pol II ChIP-seq read coverage between every pair of biological replicates. E) Principal Component Analysis (PCA) of gene-body Pol II coverage in 11,942 Pol II-bound protein-coding genes. F) Cumulative fraction of 1,759 Pol II-lost genes and all other genes (y-axis) along the scale of differential gene expression between Lmna CKO hearts and wild-type hearts (x-axis). P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes. G) Gene expression state of Pol II-lost genes, Pol II-gained genes, and all other genes in the cardiomyocyte population in Lmna CKO (n=3) versus WT (n=3) hearts derived from single-nucleus RNA-seq in En et al. 2024. P, DESeq2 p -value. H) Same as F, but along the scale of differential gene expression between Lmna CKO and wild-type pseudo-bulk cardiomyocytes from the single-nucleus RNA-seq. P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes.

Article Snippet: We used AAVpro 293T cells (TaKaRa, 632273) for MyoAAV production and BJ-5ta cells (human hTERT-immortalized foreskin fibroblast; ATCC, CRL-4001) for ChIP-seq spike-in controls.

Techniques: ChIP-sequencing, Sequencing, Control, Derivative Assay, Gene Expression, RNA Sequencing

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