Journal: eLife

Article Title: STAT3 promotes RNA polymerase III-directed transcription by controlling the miR-106a-5p/TP73 axis

doi: 10.7554/eLife.82826

Figure Lengend Snippet: ( A–C ) STAT3 knockdown reduced the synthesis of Pol III products in HepG2 cells. HepG2 cell lines stably expressing STAT3 shRNA or control shRNA were generated by a lentiviral transduction system. STAT3 expression was analyzed by RT-quantitative PCR (qPCR) ( A ) and western blot ( B ). Pol III products were monitored by RT-qPCR ( C ). ( D–F ) STAT3 knockdown decreased the synthesis of Pol III products in 293T cells. 293T cell lines stably expressing STAT3 shRNA or control shRNA were generated as described in A and B. STAT3 expression ( D and E ) and Pol III products ( F ) were detected as described in A–C. ( G and H ) STAT3 overexpression activated the expression of Pol III products in HepG2 cells. A HepG2 cell line stably expressing mCherry-STAT3 shRNA and its control cell line was generated by a lentiviral transduction system. STAT3 protein and Pol III products were analyzed by western blot ( G ) and RT-qPCR ( H ), respectively. ( I and J ) STAT3 overexpression enhanced the expression of Pol III products in 293 cells. A 293T cell line stably expressing mCherry-STAT3 shRNA and its control cell line were generated using a lentiviral transduction system. STAT3 protein and Pol III products were detected by western blot ( I ) and RT-qPCR ( J ), respectively. ( K and L ) STAT3 silencing inhibited the expression of tRNA genes. The expression of tRNA genes randomly selected was monitored by RT-qPCR using HepG2 ( K ) and 293T ( L ) cell lines with STAT3 depletion. ( M and N ) STAT3 overexpression activated the expression of tRNA genes. HepG2 ( M ) and 293T ( N ) cell lines with STAT3 overexpression were used to analyze the expression of tRNA genes by RT-qPCR. Each column in A, C, D, F, H, and J–N represents the mean ± SD of three biological replicates (n=3). *, p<0.05; **, p<0.01. p Values were obtained by Student’s t test performed with data for the control and treated samples. Mean, SD and p values were calculated using GraphPad Prism 8 software. Figure 1—source data 1. Raw images for Western blot data in . Figure 1—source data 2. Original digital data in .

Article Snippet: STAT3 cDNA was synthesized using the total RNA extracted from 293T cells and reverse transcriptase (New England Biolabs, USA), amplified by PCR, and inserted immediately downstream of a mCherry gene in the protein-expressing lentiviral plasmid pLV-EF1α-mCherry-puro.

Techniques: Stable Transfection, Expressing, shRNA, Generated, Transduction, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Over Expression, Software

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A) Experimental scheme. Cochlear epithelial cells from P5 Atoh1-BFP transgenic mice were infected with mCherry expressing control (Ctrl) virus, or virus that co-expressed mCherry and full-length (TRIM71) or mutant TRIM71 protein (Δ RING, Δ Coiled Coil, Δ NHL) (B-C) Colony forming efficiency (B) (n=4, three independent experiments) and organoid diameter in (C) (n=4, three independent experiments) after 10 days of expansion. ( D ) Cell proliferation in control and TRIM71 -expressing organoids. An EdU pulse was given at day 8 and EdU incorporation (red) was analyzed 1.5 hours later. JAG1 (green) marks supporting cells/prosensory cells, mCherry (red) marks infected cells, Hoechst labels cell nuclei (blue). ( E ) Percentage of EdU + mCherry + cells per organoids in (D) (n = 5, two independent experiments). (F) Bright field (BF), red (mCherry) and green (Atoh1-nGFP) fluorescent images of organoid cultures. (C) Percentage of Atoh1-nGFP + mCherry + organoids in (B) (n=5, three independent experiments). (D) RT-qPCR of Atoh1 mRNA in organoids (n=3, two independent experiments). (E-G) Confocal images of MYO7A immuno-stained (magenta) organoids Atoh1-GFP (green) and MYO7A (magenta) marks nascent hair cells. MCherry (red) marks infected cells. (F) Quantification of MYO7A + mCherry + cells per organoid in (E). (G) RT-PCR of Myo7a mRNA expression in organoids. Individual data points represent the average value per animal. P-values were calculated using one-way ANOVA with Tukey’s correction with the exception of (E) were two-tailed, unpaired t test was used. * P ≤ 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Transgenic Assay, Infection, Expressing, Mutagenesis, Quantitative RT-PCR, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A) Schematic of Trim71 conditional and knockout allele. (B) Experimental scheme. Cochlear epithelial cells from stage P2 TetO-Cre;R26 rtTA*M2 ;Trim71 f/f mice ( Trim71 KO) and littermates that lacked TetO-Cre transgene (WT) were cultured in the presence of doxycycline (dox). (C) Bright field (BF) images of organoid culture after 6 days of expansion. (D) Colony forming efficiency in (C). (E) Organoid diameter in (C) (n=4 in WT, n=5 in Trim71 KO, two independent experiments). (F) Confocal images of EdU (red) and Hoechst (blue) labeled organoids. A single EdU pulse was given at day 7 and EdU incorporation was analyzed 1.5 hours later. (G) Percentage of EdU + cells in (F) (n=15 in WT, n=11 in Trim71 KO, three independent experiments). (H) Experimental scheme. (I) RT-qPCR of Trim71 and progenitor ( Hmag2 and Fat3 ) and hair cell-specific ( Atoh1, Gfi1 and Pou4f3 ) mRNAs in control and Trim71 KO organoids at day 10 (n=4 in WT, n=5 in Trim71 KO, two independent experiments). (J) RT-qPCR of Trim71 and hair cell-specific ( Atoh1, Gfi1 and Pou4f3 ) mRNAs in WT and Trim71 KO organoids after 2 days of differentiation (n=3, two independent experiments). (K) Confocal images of MYO7A and SOX2 immuno-stained organoids after 6 days of differentiation. Nascent hair cells co-express Atoh1-nGFP (green), MYO7A (red) and SOX2 (magenta). Two-tailed, unpaired t test was used to calculate P values. * P ≤ 0.05, ** P < 0.01 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Knock-Out, Cell Culture, Labeling, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A) Schematic of experimental strategy. Organoid cultures were established with cochlear epithelial cells from stage E13.5 Trim71 KO ( TetO-Cre; R26rtTA*M2; Trim71f/f; Atoh1-nGFP ) mice and littermates that lacked TetO-Cre transgene (WT). Timed pregnant dam received doxycycline (dox) containing feed starting at E5.5 until tissue harvest. The Atoh1-nGFP expression marks nascent hair cells. (B) Bright field (BF) images of E13.5 wild type and Trim71 KO organoids at 7 and 10 days of expansion. (C) Organoid diameter in (B) (n=3, two independent experiments). (D) Colony forming efficiency in (B) (n=3, two independent experiments). (E) Cell proliferation in E13.5 wild type and Trim71 KO organoids. An EdU pulse was given at 7 days of expansion and EdU incorporation (red) was analyzed 1.5 hours later. Hoechst labels cell nuclei (blue). (F) Percentage of EdU + cells in E13.5 wild type (blue) and Trim71 KO (red) organoids (n = 9, three independent experiments). (G) Low power bright field (BF) and green fluorescent (Atoh1-nGFP) images of E13.5 wild type and Trim71 KO organoid cultures. (H) Quantification of Atoh1-nGFP + organoids in (G) (n=3, two independent experiments). Individual date points represent the average value per animal. Two-tailed, unpaired t test was used to calculate P values in (C), (D), (F) and (H). ** P < 0.01 and *** P < 0.001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A-C) Bulk RNA sequencing was used to analyze gene expression in P5 control and TRIM71 -expressing cochlear organoids at 10 days of expansion. ( A ) Volcano plot of RNA-seq data. Plotted is beta-value (x-axis) versus −log10 q-value (y-axis). Transcripts that are significantly upregulated in response to TRIM71 expression are marked in red circles, and transcripts that are significantly downregulated are marked in blue circles. ( B ) Biological processes and pathways associated with TRIM71-upregulated genes ranked by adjusted p-value (q-value). ( C ) Biological processes and pathways associated with TRIM71-downregulated genes ranked by adjusted p-value (q-value). ( D ) Venn diagram showing intersection between TRIM71-upregulated genes, predicted let-7 target genes and LIN28B+FST-upregulated genes. (E) RT-qPCR of Hmga2 mRNA expression in P5 control (Ctrl) cochlear organoids and cochlear organoids that expressed full length ( TRIM71 ), RING deficient (Δ RING ), Coiled-Coil deficient ( ΔCoiled-Coil ) or NHL deficie n t ( ΔNHL ) TRIM71 protein at 10 days of expansion (n=3, two independent experiments). (F) TaqMan assay of mature let-7a-5p, let-7d-5p, let-7g-5p, let-7i-5p transcripts in P5 control (Ctrl) and TRIM71 expressing cochlear organoids (n=3, two independent experiments). (G ) RT-qPCR-based analysis of Bmp4, Id1, Id2, Id3 expression (n=3, two independent experiments) in control, TRIM71 or TRIM71 ΔNHL expressing cochlear organoids after 10 days of expansion (n=3, two independent experiments). Individual data points represent the average value per animal. One-way ANOVA with Tukey’s correction was used to calculate P values. * P ≤ 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, TaqMan Assay

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A) TaqMan assay of mature let-7a-5p, let-7d-5p, let-7g-5p, let-7i-5p transcripts in P5 control (Ctrl) and LIN28B -expressing cochlear organoids (n=3, two independent experiments). Two-tailed, unpaired t test was used to calculate P values. (B) Immunoblots were used to analyze endogenous LIN28B, AGO2, HMGA2, P-SMAD1/5/9 and β-actin protein levels in P5 control, TRIM71 and ΔNHL expressing cochlear organoids at 10 days of expansion. (C) RT-qPCR of Bmp4 mRNA expression (n=3, two independent experiments) in P5 control (Ctrl) cochlear organoids and cochlear organoids that expressed full length ( TRIM71 ), RING deficient (Δ RING ), Coiled-Coil deficient ( ΔCoiled-Coil ) or NHL deficient ( ΔNHL ) TRIM71 protein at 10 days of expansion. Individual date points represent the average value per animal. One-way ANOVA with Tukey’s correction was used to calculate P values. ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: TaqMan Assay, Expressing, Two Tailed Test, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A) Confocal images showing JAG1 (magenta) and HMGA2 (green) expression in P5 control and TRIM71 -expressing cochlear organoids at 10 days of expansion. (B) Quantification of JAG1 + mCherry + HMGA2 + cells per organoid in (A) (n=3, two independent experiments). (C) Confocal images showing JAG1 (magenta) and S100A1 (green) expression in P5 control and TRIM71- expressing cochlear organoids at 10 days of expansion. (D) Quantification of JAG1 + mCherry + S100A1 + cells per organoid in (A) (n=3, two independent experiments). (E) Confocal images showing NFIB (magenta) and Atoh1-nGFP (green) expression in P5 control and TRIM71- expressing cochlear organoids after 2 days in differentiation. (F) Quantification of NFIB low , NFIB high , NFIB low Atoh1-nGFP + , NFIB high Atoh1-nGFP + cells in control and TRIM71 -expressing organoids (n=4, two independent experiments). Individual data points represent the average value per animal. One-way ANOVA with Tukey’s correction was used to calculate P values in (B) and (D). Two-way ANOVA with Tukey’s correction was used to calculate P values in (F). * P ≤ 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Expressing

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A-B) Cochlear ZBTB20 and NFIB protein expression in vivo. Confocal images of the hair cell layer (HC) and supporting cell layer (SC) of cochlear sensory of stage P5 mice. SOX2 (green) marks supporting cells (SC) and Kölliker’s cells (KC) but not Claudius cells (CC). Atoh1-nGFP (green) marks hair cells (HC). (A) NFIB (magenta) is expressed in Claudius cells, supporting cells and Kölliker’s cells but not in hair cells. (B) ZBTB20 (magenta) is expressed in Claudius cells and supporting cells, but not in Kölliker’s cells or hair cells. (C) Confocal images of ZBTB20 protein (magenta) and Atoh1-nGFP transgene (green) expression in P5 control and TRIM71 expressing cochlear organoids after 2 days of differentiation. (D) Quantification of ZBTB20 low , ZBTB20 hi g h , ZBTB20 low Atoh1-nGFP + , ZBTB20 hi g h Atoh1-nGFP + cells in (C) (n=4, two independent experiments). Individual date points represent the average value per animal. Two-way ANOVA with Tukey’s correction was used to calculate P values in D. *P ≤ 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Expressing, In Vivo

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A-E) LIN28B enhances TRIM71’s positive effect on supporting cell reprogramming and hair cell formation. (A) Experimental scheme. (B) Bright field (BF) and red and green fluorescent images of P5 cochlear organoid cultures infected with control (Ctrl) virus, or virus that expressed mouse Lin28b , human TRIM71 or both ( Lin28b+TRIM71 ) at 2 days of differentiation. MCherry (red) marks infected cells and Atoh1-nGFP (green) marks nascent hair cells. (C) Percentage of mCherry + Atoh1-nGFP + organoids in (B). (D-E) RT-qPCR-analysis of progenitor ( Hmga2 ) (D) and supporting cell-specific ( Zbtb20, Nfib ) (E) gene expression in control, Lin28b, TRIM71 or Lin28b+ TRIM71- expressing organoids after 10 days of expansion. Individual date points represent the average value per animal. (F-K) Loss of Hmga2 diminishes the mitotic and hair cell-forming potential of cochlear supporting cells/Kölliker’s cells. ( F ) Experimental scheme. ( G ) Colony forming efficiency in Hmga2 knockdown and control cultures at 8 days in vitro (DIV). ( H ) Organoid diameter in Hmga2 knockdown and control cultures at 8 DIV. ( I ) RT-qPCR analysis of hair cell ( Aoth1 and Pou4f3 ) and pro-sensory-specific ( Trim71 ) gene expression (n=3, two independent experiments). Hmga2 expression was analyzed to confirm knockdown. ( J ) Low power Bright field (BF) and green fluorescent images (Atoh1-nGFP) of Hmga2 knockdown and control cultures. ( K ) Quantification of Atoh1-nGFP + organoids in (J). Two-way with Tukey’s correction was used to calculate P values in (C-E) and one-way ANOVA with Tukey’s correction was used to calculate P values in (J), (H), (I) and (K). * P ≤ 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Infection, Quantitative RT-PCR, Expressing, In Vitro

Journal: bioRxiv

Article Title: Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

doi: 10.1101/2023.01.12.523802

Figure Lengend Snippet: (A) Schematic of lentiviral expression cassettes used to express human TRIM71 and mouse Lin28b in P5 cochlear organoid cultures. (B) RT-qPCR-analysis of Nfia, Nfic and Nfix expression in control (Ctrl), Lin28b, TRIM71 or Lin28b+TRIM71-expressing organoids after 10 days of expansion. Two-way ANOVA with Tukey’s correction was used to calculate P values. * P ≤ 0.05,

Article Snippet: TRIM71-R608H expression plasmid was generated by cloning DNA fragment-R608H (Integrated DNA Technology) (table S5) into pCDH-EF1a-3xHA-TRIM71-T2A-mCherry plasmid using SmaI (NEB, no. R0141) and EcoRI-HF (NEB, no. R3101).

Techniques: Expressing, Quantitative RT-PCR

Journal: Nanotheranostics

Article Title: Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?

doi: 10.7150/ntno.23826

Figure Lengend Snippet: SSTR2, 3 and 5 expression in cell lines employed in the study: indirect immunolabelling in a flow cytometry analysis. SSTR2, 3 and 5 immunolabelling results in paraformaldehyde (PFA)-fixed and saponin-permeabilized HEK293 and BON1 cells, along with matched control stains in viable non-permeabilized HEK293 cells are presented on panels A and B, respectively. As all the anti-SSTR antibodies (Abs) employed in the series on panel A target native epitopes within C -tails of the receptors (confined to cytoplasmic compartment), the cells were fixed with PFA and permeabilized with saponin before immunolabelling. Conversely, the immunolabelling of the cells on panel B involved primary Ab against distinct tags within extracellular N -termini of SSTRs, hence no permeabilization was required and the staining was done on viable non-permeabilized cells. Noteworthy, the pattern of signal from matched samples stained for the same target with Abs against its different epitopes (Abs to intracellular C -tails of receptors on panel A vs Abs to tags within extracellular domains of the same receptors on panel B) is almost identical, which signifies specificity of the data. Bimodal appearance of the populations on histograms, especially obvious in case of SSTR3- and 5-overexpressing cells, is explained by the oligoclonal nature of the cultures, with the resulting distribution being formed by progeny of two (or more) dominant clones. Staining for β-tubulin, a component of a cytoskeleton, on panels A and B was implemented as a positive or negative control of permeabilization, respectively. The cells were analyzed on LSRII cytometer; at least 15 000 of the gated events were captured. Every image represents an overlay histogram of two samples: black transparent charts stand for either non-stained controls or fully stained samples; shaded green charts reflect the corresponding secondary antibody - only stained controls. x-axis denotes sample emission [(505 nm longpass)/(530/30 nm bandpass)] upon stimulation with 488 nm laser; y-axis indicates the number of events registered. The data from a single representative experiment (performed in duplicate) is shown; the complete series has been independently performed at least three times.

Article Snippet: For the assembly of SSTR3_Myc-P2A-mCherry plasmid, pMiniT-SSTR3_Myc vector was double digested with BsiWI (New England Biolabs, Cat# R0553) and BssHII (Promega, Cat# R6831) and the released SSTR3_Myc coding sequence was gel-purified.

Techniques: Expressing, Flow Cytometry, Staining, Clone Assay, Negative Control, Cytometry