Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: MicroRNA-dependent disruption of splice-relevant stem–loops controls Fas3 exon 5 inclusion in Drosophila . ( A ) Schematic of Fas3 pre-mRNA showing box I (red) and box II (blue), which form a splice-relevant stem surrounding exon 5. ( B ) Table listing microRNAs predicted to bind box regions with their corresponding ΔG MIMOSAS values; energetically (un)favorable candidates selected for downstream experiments are highlighted. ( C ) Model of MIMOSAS-mediated alternative splicing of Fas3 . When highlighted microRNAs disrupt stem formation, the RC splice variant that includes exon 5 is produced. In the absence of MIMOSAS activity, alternative Fas3 splice variants lacking exon 5 (RA/B/D/E/F/G) predominate. ( D, E ) ΔG MIMOSAS values and corresponding binding probabilities for miR-973 and miR-976 at box I, and miR-1000 and miR-999 at box II. microRNA binding probability was calculated for a 200-nucleotide nascent pre-mRNA segment transcribed from the 5′ end by RNA polymerase. Three colors indicate distinct transcriptional stages of the box: green, when RNA polymerase has just reached the 5′ end of the box; orange, when the 3′ end of the box has just been transcribed; and dark red, when an additional downstream segment equal in length to the box has been transcribed. Ratios of endogenous RA/B/D/E/F/G (STEM) variants to total Fas3 mRNA measured by real-time PCR in brains overexpressing microRNAs ( F ) or expressing microRNA sponges for knockdown ( G ) under the pan-neuronal driver elav C155 -GAL4 , and in a miR-999 KO loss-of-function mutant background ( H ). Ratios of STEM to Total in miR-9c and scramble sponge-expressing brains, as well as wild-type ( w 1118 ) control groups were normalized to 1, and fold changes are shown. Data are presented as mean ± s.d. Statistical significance was determined using unpaired Student’s t- test, **** P ≤ .0001, *** P ≤ .001; n ≥ 3, triplicate sampling.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Disruption, Alternative Splicing, Variant Assay, Produced, Activity Assay, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Mutagenesis, Control, Sampling

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: MicroRNA-mediated regulation of RpL3 splice variant expression in the Drosophila brain. ( A ) Schematic of the RpL3 gene and its predicted splice variants. Color-coded boxes I, II, and III denote sequences that form stem–loop structures required for alternative splicing. Several microRNAs predicted to bind boxes I and II are indicated, with the potential to bias production of specific splice isoforms. The black arrow marks the shared donor splice site, while purple (proximal) and green (distal) arrows indicate the two acceptor splice sites. Locations of three TaqMan real-time PCR probes used in subsequent experiments are shown: total RpL3 mRNA (black), RA/RH plus RG (brown), and RG only (purple). ( B ) Pie charts showing the relative proportions of endogenous RpL3 splice variants in adult wild-type fly heads (yw), as determined by RT-PCR . Scatter plots showing total RpL3 mRNA levels and the relative abundance of individual RpL3 splice variants, quantified by two-color multiplex real-time PCR with variant-specific TaqMan probes. Measurements were obtained from fly brains overexpressing microRNAs under the pan-neuronal driver elav C155 -GAL4 ( C–F ) or from miR-210 KO and miR-988 KO loss-of-function mutants ( G–J ). The abundances of RA/RH and RD were inferred from the difference between total RpL3 and the sum of the directly measured variants. Values for the miR-9c overexpression group and wild-type ( w 1118 ) flies were normalized to 1, and fold changes are shown. rp49 served as an internal control. Data are presented as mean ± s.d. Statistical significance was assessed by one-way ANOVA with Bonferroni’s post hoc test ( C–F ) or unpaired Student’s t -test ( G–J ), *** P ≤ .001; n = 4, triplicate sampling.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Variant Assay, Expressing, Alternative Splicing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay, Over Expression, Control, Sampling

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: MicroRNA-mediated regulation of RpL3 alternative splicing uncovered using a dual-color split-fluorescent reporter. ( A ) Schematic of the RpL3 splicing reporter construct, incorporating key pre-mRNA sequences essential for STEM formation, self-complementing split-fluorescent proteins (FPs), and nuclear (NLS), or cytoplasmic (NES) localization signals. The construct models three distinct splicing outcomes, each corresponding to a specific mRNA variant and resulting protein product, alongside its associated visualization strategy. ( B ) Predicted secondary structure of the RpL3 ASR generated by FORNA depicts an MFE model, showing stem formation between box I (pink) and box II (cyan), as well as between box II (cyan) and box III (yellow). MFE values for each stem were computed using RNAcofold from the ViennaRNA Package . ( C ) Analysis of nucleotide base-pairing interactions between wild-type microRNAs and box sequences, with MFE values calculated by RNAcofold . Box sequences are highlighted along the y-axis (pink for box I, cyan for box II), while microRNA sequences are displayed along the x -axis. ( D ) Table indicating nucleotide base-pairing interactions between microRNAs and box sequences. Scrambled mutant variants are marked in red. MFE values (kcal/mol) for each microRNA duplex, calculated by RNAcofold , are color-coded between interacting sequence pairs.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Alternative Splicing, Construct, Variant Assay, Generated, Mutagenesis, Sequencing

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: Visualization and quantification of microRNA-mediated RpL3 splicing outcomes in COS-7 cells. ( A – D ) Representative confocal microscopy images of COS-7 cells transfected with the RpL3 splicing reporter and sfCherry11-pre-microRNA constructs (wild type and mutant). Images were acquired 48 h post-transfection. Nuclei are marked with DAPI (blue) and Lamin A/C (white); fluorescence intensity is depicted using heat maps. Scale bar: 10 μm. ( A’–D’ ) Violin plots summarizing fluorescence ratios calculated from panels (A–D). Cytoplasmic GFP/total fluorescence (GFP + mCherry) indicates RA/H variant (green dots), nuclear GFP/total fluorescence depicts RG variant (purple dots), and mCherry/total fluorescence reflects RD variant (red dots). Data represent median ± quartiles ( n ≥ 25 cells per group). Statistical comparisons were performed using one-way ANOVA with Bonferroni’s post hoc test; distinct letters denote statistically different groups ( P ≤ .05). ( A’’–D’’ ) Schematic diagrams illustrating the effects of each microRNA on the splicing reporter are shown beneath their respective columns of cell images.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Confocal Microscopy, Transfection, Construct, Mutagenesis, Fluorescence, Variant Assay

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: Nuclear microRNAs drive MIMOSAS in an AGO1-dependent manner. ( A ) Subcellular localization of AGO1 in larval salivary gland cells with or without miR-210 pre-microRNA expression in motor neurons ( vGlut OK371 -GAL4 ) in wild-type ( AGO1 +/+ ) and heterozygous ( AGO1 +/− ) backgrounds. Nuclei are labeled with DAPI (blue), AGO1 with immunostaining (green). Yellow dashed lines outline individual salivary gland cells. White boxes highlight single cells, with corresponding intensity heat maps shown below each group. Scale bar, 20 μm. ( B ) Violin plots showing the percentage of nuclear AGO1. AGO1 fluorescence intensity was quantified using ImageJ. For each experiment, 8–13 salivary gland cells per larva and ≥4 larvae per group were analyzed. Data are presented as median ± quartiles. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test; distinct letters indicate groups that differ significantly ( P ≤ .05). ( C ) Box-and-whisker plots of average AGO1 intensity per salivary gland, with all data points displayed (min to max). At least four larvae per group were analyzed. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test; distinct letters indicate statistically different groups ( P ≤ .05). ( D ) Subcellular localization of miR-210-3p in larval salivary gland cells expressing miR-210 pre-microRNA in motor neurons ( vGlut OK371 -GAL4 ) in AGO1 +/+ and AGO1 +/− backgrounds. Nuclei are labeled with DAPI (blue), and miR-210-3p is detected by antisense FISH probe (gray), with signal intensities visualized as heat maps. Scale bar, 20 μm. ( E ) Violin plots summarizing the percentage of nuclear miR-210-3p. Fluorescence intensity was quantified using ImageJ. For each experiment, 13–16 salivary gland cells per larva and ≥6 larvae per group were analyzed. Data are shown as median ± quartiles. Statistical significance was determined by unpaired Student’s t -test, **** P ≤ .0001. ( F ) Box-and-whisker plots of average miR-210-3p intensity per salivary gland, with all data points shown (min to max). At least six larvae per group were measured. Statistical comparisons were performed using an unpaired Student’s t-test; ns, not significant. Scatter plots showing the ratios of endogenous Nmnat-RA (STEM) to Nmnat-RB (MIMOSAS) ( G ) and RpL3 RG splice variant to total RpL3 ( H ), quantified by two-color multiplex real-time PCR with variant-specific TaqMan probes in fly brains overexpressing microRNAs under the pan-neuronal driver elav C155 -GAL4 in AGO1 +/+ and AGO1 +/− backgrounds. Ratios in the miR-9c–expressing AGO1 +/+ group were set to 1, and fold changes are shown. Data are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post hoc test; **** P ≤ .0001, ** P ≤ .01; n = 3, triplicate sampling.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Expressing, Labeling, Immunostaining, Fluorescence, Whisker Assay, Variant Assay, Multiplex Assay, Real-time Polymerase Chain Reaction, Sampling

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: MicroRNA-mediated regulation of RpL3 splice variant expression in the Drosophila brain. ( A ) Schematic of the RpL3 gene and its predicted splice variants. Color-coded boxes I, II, and III denote sequences that form stem–loop structures required for alternative splicing. Several microRNAs predicted to bind boxes I and II are indicated, with the potential to bias production of specific splice isoforms. The black arrow marks the shared donor splice site, while purple (proximal) and green (distal) arrows indicate the two acceptor splice sites. Locations of three TaqMan real-time PCR probes used in subsequent experiments are shown: total RpL3 mRNA (black), RA/RH plus RG (brown), and RG only (purple). ( B ) Pie charts showing the relative proportions of endogenous RpL3 splice variants in adult wild-type fly heads (yw), as determined by RT-PCR . Scatter plots showing total RpL3 mRNA levels and the relative abundance of individual RpL3 splice variants, quantified by two-color multiplex real-time PCR with variant-specific TaqMan probes. Measurements were obtained from fly brains overexpressing microRNAs under the pan-neuronal driver elav C155 -GAL4 ( C–F ) or from miR-210 KO and miR-988 KO loss-of-function mutants ( G–J ). The abundances of RA/RH and RD were inferred from the difference between total RpL3 and the sum of the directly measured variants. Values for the miR-9c overexpression group and wild-type ( w 1118 ) flies were normalized to 1, and fold changes are shown. rp49 served as an internal control. Data are presented as mean ± s.d. Statistical significance was assessed by one-way ANOVA with Bonferroni’s post hoc test ( C–F ) or unpaired Student’s t -test ( G–J ), *** P ≤ .001; n = 4, triplicate sampling.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Variant Assay, Expressing, Alternative Splicing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay, Over Expression, Control, Sampling

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: Multiple microRNAs regulate Nmnat splice variants via MIMOSAS. ( A ) Schematic of the Nmnat gene showing a pair of boxes flanking exon 5 that are targeted by several microRNAs. Stem formation between these boxes favors the production of the splice variant RA, whereas MIMOSAS activity promotes the RB variant. ( B ) Table of microRNAs predicted to bind the box regions with their corresponding ΔG MIMOSAS values. microRNAs are ordered by their binding characteristics to box I and box II; highlighted candidates were selected for experimental validation. Ratios of endogenous RA (STEM) to RB (MIMOSAS) variants measured by real-time PCR in brains overexpressing microRNAs ( C ), expressing microRNA sponges for knockdown ( D ) under the pan-neuronal driver elav C155 -GAL4 , or in miR-137 KO , miR-210 KO , and miR-1002 KO loss-of-function mutants ( E ). Ratios of RA to RB in miR-9c-expressing and scramble sponge controls, as well as wild-type ( w 1118 ) flies, were normalized to 1, and fold changes are shown. Data are presented as mean ± s.d. Statistical significance was determined using unpaired Student’s t -test, **** P ≤ .0001, *** P ≤ .001, ** P ≤ .01, * P ≤ .05; n ≥ 3, triplicate sampling. ( F ) Schematic of the Nmnat ASR. EGFP fluorescence reports production of the RB splice variant (MIMOSAS), whereas mCherry fluorescence indicates the RA splice variant (STEM). ( G ) Confocal images of pupal brains (95 h after pupal formation, APF) co-expressing the ASR and microRNAs under nysb-GAL4 . Nuclei are labeled with DAPI (white), and mCherry and EGFP intensities are visualized with heat maps. Scale bar is 50 μm. ( H ) Scatter plot of integrated mCherry and EGFP fluorescence intensities. Data are mean ± s.d. ( n = 10 brains per group). Statistical significance was assessed by one-way ANOVA with Bonferroni’s post hoc test, *** P ≤ .001, ** P ≤ .01, * P ≤ .05. ( I ) Violin plot of the mCherry-to-EGFP intensity ratio. Data are shown as median ± quartiles ( n = 10 brains per group). Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test compared with the miR-9c-expressing control; groups with different letters differ significantly ( P ≤ .05).

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Variant Assay, Activity Assay, Binding Assay, Biomarker Discovery, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Sampling, Fluorescence, Labeling, Control

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: miR-210 promotes MIMOSAS splicing by binding intronic box I in a sequence-specific manner. ( A ) In silico prediction of interactions between miR-210 variants and intronic box I. A FORNA MFE model of the Nmnat ASR shows a stem formed between box I (cyan) and box II (pink). Wild-type miR-210-3p (yellow) and scrambled mutant variants (red) are indicated. MFE values (kcal/mol) for each miR-210-box I duplex were calculated using the duplexfold function in RNAstructure and are color-coded between interacting sequence pairs. ( B ) Schematic of the Nmnat splicing reporter, which incorporates key pre-mRNA elements required for STEM formation, a split-GFP cassette, and either nuclear localization signal (NLS) or nuclear export signal (NES) tags. Two alternative splicing outcomes are illustrated, along with their corresponding mRNA isoforms, protein products, and fluorescence readouts. ( C ) Representative images of HEK293T cells co-transfected with the Nmnat splicing reporter and sfCherry2-tagged pre-microRNAs (miR-9c, miR-210WT, miR-210MT1, miR-210MT2, or miR-210MT3). Cells were imaged 48 h post-transfection. Nuclei are labeled with DAPI (blue), and GFP intensity is shown in grayscale. The red dot circles the nuclear envelope. Scale bar: 10 μm. ( D ) Scatter plot quantifying the STEM-to-MIMOSAS splice variant ratio by real-time PCR in HEK293T cells co-transfected with the ASR and the indicated microRNAs. The miR-9c control group was normalized to 1. Data are presented as mean ± s.d. from n ≥ 4 biological replicates (triplicates per group). Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test; groups with different letters differ significantly ( P ≤ .05). ( E ) Violin plot quantifying the cytoplasmic-to-total GFP intensity ratio from panel (C). Data are shown as median ± quartiles ( n ≥ 40 cells per group). Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc test; groups labeled with different letters are significantly different ( P ≤ .05). ( F ) Logarithmic regression of MIMOSAS output as a function of wild-type miR-210 expression, based on the percentage of cytoplasmic GFP intensity and the mCherry-to-total GFP intensity ratio. Data points highlighted in blue (value < 2 8 ) were included in the fit; grey points were excluded due to a plateau effect.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Binding Assay, Sequencing, In Silico, Mutagenesis, Alternative Splicing, Fluorescence, Transfection, Labeling, Variant Assay, Real-time Polymerase Chain Reaction, Control, Expressing

Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: Nuclear microRNAs drive MIMOSAS in an AGO1-dependent manner. ( A ) Subcellular localization of AGO1 in larval salivary gland cells with or without miR-210 pre-microRNA expression in motor neurons ( vGlut OK371 -GAL4 ) in wild-type ( AGO1 +/+ ) and heterozygous ( AGO1 +/− ) backgrounds. Nuclei are labeled with DAPI (blue), AGO1 with immunostaining (green). Yellow dashed lines outline individual salivary gland cells. White boxes highlight single cells, with corresponding intensity heat maps shown below each group. Scale bar, 20 μm. ( B ) Violin plots showing the percentage of nuclear AGO1. AGO1 fluorescence intensity was quantified using ImageJ. For each experiment, 8–13 salivary gland cells per larva and ≥4 larvae per group were analyzed. Data are presented as median ± quartiles. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test; distinct letters indicate groups that differ significantly ( P ≤ .05). ( C ) Box-and-whisker plots of average AGO1 intensity per salivary gland, with all data points displayed (min to max). At least four larvae per group were analyzed. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test; distinct letters indicate statistically different groups ( P ≤ .05). ( D ) Subcellular localization of miR-210-3p in larval salivary gland cells expressing miR-210 pre-microRNA in motor neurons ( vGlut OK371 -GAL4 ) in AGO1 +/+ and AGO1 +/− backgrounds. Nuclei are labeled with DAPI (blue), and miR-210-3p is detected by antisense FISH probe (gray), with signal intensities visualized as heat maps. Scale bar, 20 μm. ( E ) Violin plots summarizing the percentage of nuclear miR-210-3p. Fluorescence intensity was quantified using ImageJ. For each experiment, 13–16 salivary gland cells per larva and ≥6 larvae per group were analyzed. Data are shown as median ± quartiles. Statistical significance was determined by unpaired Student’s t -test, **** P ≤ .0001. ( F ) Box-and-whisker plots of average miR-210-3p intensity per salivary gland, with all data points shown (min to max). At least six larvae per group were measured. Statistical comparisons were performed using an unpaired Student’s t-test; ns, not significant. Scatter plots showing the ratios of endogenous Nmnat-RA (STEM) to Nmnat-RB (MIMOSAS) ( G ) and RpL3 RG splice variant to total RpL3 ( H ), quantified by two-color multiplex real-time PCR with variant-specific TaqMan probes in fly brains overexpressing microRNAs under the pan-neuronal driver elav C155 -GAL4 in AGO1 +/+ and AGO1 +/− backgrounds. Ratios in the miR-9c–expressing AGO1 +/+ group were set to 1, and fold changes are shown. Data are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post hoc test; **** P ≤ .0001, ** P ≤ .01; n = 3, triplicate sampling.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Expressing, Labeling, Immunostaining, Fluorescence, Whisker Assay, Variant Assay, Multiplex Assay, Real-time Polymerase Chain Reaction, Sampling

Journal: bioRxiv

Article Title: Comparison studies between Cesium-137 and X-ray irradiators in epithelial injury using in vitro and in vivo models

doi: 10.64898/2026.04.17.719248

Figure Lengend Snippet: RT-qPCR analysis of CDKN1A ( a, f, k ), KLF5 ( b, g, l ), KLF4 ( c, h, m ), BAX ( d, i, n ), and BCL2 ( e, j, o ) in HCT116, RKO and DLD-1 calculated as a fold change at 6, 24, 48 and 72 h after irradiation. HPRT1 was used as a housekeeping gene (control). RT-qPCR was performed as described in the “Materials and methods” section. Data points represent the average of three independent experiments, with the mean ±SD indicated. Significance was determined by the Student’s test followed by an analysis of the normal distribution (Tukey’s test), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Commercially available TaqMan primers detecting mouse ChgA (Mm00514341-FAM), Alpi1 (Mm01285814-FAM), Olfm4 (Mm01320260-FAM), Cdkn1a (Mm00432448-FAM), Lgr5 (Mm00438890-FAM), Mki67 (Mm01278617-FAM), Actb (Mm04394036-VIC) and human CDKN1A (Hs00355782-FAM), KLF4 (Hs00358836-FAM), KLF5 (Hs0000000-FAM), BCL2 (Hs01048932-FAM), BAX (Hs00180269-FAM) and HPRT1 (Hs02800695-VIC) transcripts were used.

Techniques: Quantitative RT-PCR, Irradiation, Control

Journal: bioRxiv

Article Title: Comparison studies between Cesium-137 and X-ray irradiators in epithelial injury using in vitro and in vivo models

doi: 10.64898/2026.04.17.719248

Figure Lengend Snippet: RT-qPCR analysis of ( a ) Cdkn1a expression levels after 6Gy, ( b ) Cdkn1a expression levels after 8Gy, ( c ) Mki67 expression levels after 6Gy, ( d ) Mki67 expression levels after 8Gy, ( e ) Lgr5 expression levels after 6Gy, ( f ) Lgr5 expression levels after 8Gy, ( g ) Olfm4 expression levels after 6Gy, ( h ) Olfm4 expression levels after 8Gy, ( i ) Alpi1 expression levels after 6Gy, ( j ) Alpi1 expression levels after 8Gy, ( k ) Chga expression levels after 6Gy, and ( l ) Chga expression levels after 8Gy were calculated as a fold change at 6, 24, 48 and 72 h with either 137 Cs or X-ray irradiation. Actb was used as a housekeeping gene (control). Data points represent the average of three independent experiments, with the mean ±SD indicated. Significance was determined by the student’s test followed by an analysis of the normal distribution (Tukey’s test), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Commercially available TaqMan primers detecting mouse ChgA (Mm00514341-FAM), Alpi1 (Mm01285814-FAM), Olfm4 (Mm01320260-FAM), Cdkn1a (Mm00432448-FAM), Lgr5 (Mm00438890-FAM), Mki67 (Mm01278617-FAM), Actb (Mm04394036-VIC) and human CDKN1A (Hs00355782-FAM), KLF4 (Hs00358836-FAM), KLF5 (Hs0000000-FAM), BCL2 (Hs01048932-FAM), BAX (Hs00180269-FAM) and HPRT1 (Hs02800695-VIC) transcripts were used.

Techniques: Quantitative RT-PCR, Expressing, Irradiation, Control