hmw poly i c lyovec  (InvivoGen)

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

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