dsrna analogue poly i c  (InvivoGen)

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.

Article Snippet: KO of MAVS and MDA5 was validated by transfecting cells with 0.1 μg/mL dsRNA analogue poly(I:C) (tlrl-picwlv, InvivoGen) complexed with lipofectamine (Invitrogen), according to the manufacturer’s instructions, or lipofectamine alone and added to cells for 24 h at 37°C.

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

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.

Article Snippet: KO of MAVS and MDA5 was validated by transfecting cells with 0.1 μg/mL dsRNA analogue poly(I:C) (tlrl-picwlv, InvivoGen) complexed with lipofectamine (Invitrogen), according to the manufacturer’s instructions, or lipofectamine alone and added to cells for 24 h at 37°C.

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

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.

Article Snippet: KO of MAVS and MDA5 was validated by transfecting cells with 0.1 μg/mL dsRNA analogue poly(I:C) (tlrl-picwlv, InvivoGen) complexed with lipofectamine (Invitrogen), according to the manufacturer’s instructions, or lipofectamine alone and added to cells for 24 h at 37°C.

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

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: Mapping of transcripts targeted by MitoString probes on a schematic representation of mtDNA. B-C: Relative levels of mt-mRNA in MTPAP_KO cells (sg#1, #2, #3 each collected 3 times) ( A ) and fibroblasts from patients with MTPAP mutations (P2, P3, P4, P5 each collected 3 times) ( B ) compared to respective controls and quantified using MitoString. H.S.: heavy strand, L.S.: light strand. Mean ± SEM; n=3 experiments; each point corresponds to a different sample, ns indicates non significance, * p< 0.05 in multiple Mann-Whitney with Holm-Sidak comparison tests. D-E: Relative levels of non-coding mtRNA in MTPAP_KO cells (sg#1, #2, #3 each collected 3 times) ( D ) and of patients with MTPAP mutations (P2, P3, P4, P5 each collected 3 times) assessed using MitoString compared to controls. H.S. : heavy strand, L.S. : light strand. Mean ± SEM; n=3 experiments; each point corresponds to a different sample, * indicates p< 0.05 in multiple Mann-Whitney with Holm-Sidak comparison tests. F: Representative confocal microscopy images of Mito-GFP mitochondrial marker and sm-FISH probes MT-CO1 -Cy5 and rc_ MT-CO1 -Cy3 in control and MTPAP_KO cells (sg#1) from 3 experiments. Scale bar: 5µm. G-H: Quantification of the average fluorescence intensity signal per cell of single particles detected with sm-FISH probe MT-CO1 -Cy3 ( G ) and rc_ MT-CO1 -Cy3 ( H ) in control and MTPAP_KO cells (average of sg#1 and sg#2). 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. I: Representative confocal microscopy images of Mito-GFP mitochondrial marker, smFISH probes MT-CO1 -Cy3, and immunostaining of dsRNA with J2 antibody in MTPAP_KO cells from 3 experiments. Scale bar: 5 µm. J: Representative confocal microscopy images of Mito-GFP mitochondrial marker, smFISH probes rc_ MT-CO1 -Cy3, and immunostaining of dsRNA with J2 antibody in MTPAP_KO cells from 3 experiments. Scale bar: 5 µm.

Article Snippet: KO of MAVS and MDA5 was validated by transfecting cells with 0.1 μg/mL dsRNA analogue poly(I:C) (tlrl-picwlv, InvivoGen) complexed with lipofectamine (Invitrogen), according to the manufacturer’s instructions, or lipofectamine alone and added to cells for 24 h at 37°C.

Techniques: MANN-WHITNEY, Comparison, Confocal Microscopy, Marker, Control, Fluorescence, Immunostaining