Journal: iScience

Article Title: Distinct but interchangeable subpopulations of colorectal cancer cells with different growth fates and drug sensitivity

doi: 10.1016/j.isci.2023.105962

Figure Lengend Snippet:

Article Snippet: For MSI1 overexpression, human MSI1 cDNA was amplified with the primers of MSI1_BamHI-F and MSI1_stopdead_XbaI-R ( ) using the PrimeScriptTM first strand cDNA Synthesis (Takara, 6210A) and PrimeSTAR Ⓡ Max DNA Polymerase kits according to manufacturer instructions (Takara, R045A).

Techniques: Recombinant, Concentration Assay, Membrane, RNAscope, Cell Viability Assay, SYBR Green Assay, Reverse Transcription, Expressing, Microarray, Derivative Assay, Software

Journal: Life sciences

Article Title: Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

doi: 10.1016/j.lfs.2020.118974

Figure Lengend Snippet: Fig. 1. Sglt3a and Sglt3b mRNA expression is localized in the epithelium of small intestinal segments in C57BL/6J mice, but absent in the kidney. (a) RNAscope in-situ hybridization using mouse Slc5a4a (Sglt3a), and Slc5a4b (Sglt3b) anti-sense hybridization probes was performed on paraffin-embedded kidney and intestinal segment tissue sections. Negative control (B. subtilis gene dihydrodipicolinate reductase anti-sense hybridization), and positive control (Peptidylprolyl Isomerase B anti-sense hybridization) is shown for respective organs. The arrow points to positive signal in Sglt3b kidney staining. (b) Sglt3a and Sglt3b mRNA expression was deter mined by quantitative PCR in matching tissue samples. Data is normalized relative to duodenum and was analysed by one-way ANOVA (Table S1) followed by post- hoc multiple comparisons with Bonferroni correction. **P < 0.01, ***P < 0.001 compared with duodenum; ###P < 0.001 compared with jejunum.

Article Snippet: Quantitative PCR was performed using HOT FIREPol Probe qPCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia) and respective assay (SLC5A4: Hs00429527_m1, LRP10: Hs01047362_m1, RPLP0: 4326314E, 18S rRNA: 4319413E; Thermo Fisher Scientific).

Techniques: Expressing, RNAscope, In Situ Hybridization, Hybridization, Negative Control, Positive Control, Staining, Real-time Polymerase Chain Reaction

Journal: Life sciences

Article Title: Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

doi: 10.1016/j.lfs.2020.118974

Figure Lengend Snippet: Fig. 2. Confirmed epithelial localization of C57BL/6J mouse Sglt3a and Sglt3b mRNA expression in isolated intestinal epithelial cells and organoids and human SGLT3 in intestinal organoids, respectively. The mRNA expression of Sglt3a and Sglt3b in (a) ileum and intestinal epithelial cells isolated from ileum and in (b) intestinal organoids derived from intestinal segments of C57BL/6J mice. (c) Human SGLT3 mRNA expression in organoids derived from crypts of duodenum, jejunum, ileum and colon. The expression was measured by qPCR. Data is normalized relative to ileal tissue (a) or duodenum (b, c) and is presented as individual values and mean ± SEM. Data was analysed by unpaired Student’s t-test (a) and one-way ANOVA (Table S1) followed by post-hoc multiple comparisons with Bonferroni correction (b,c), respectively. †††P <0.001 compared with ileal tissue; ***P < 0.001 compared with duodenum; ###P < 0.001 compared with jejunum; §§§P < 0.001 compared with ileum.

Article Snippet: Quantitative PCR was performed using HOT FIREPol Probe qPCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia) and respective assay (SLC5A4: Hs00429527_m1, LRP10: Hs01047362_m1, RPLP0: 4326314E, 18S rRNA: 4319413E; Thermo Fisher Scientific).

Techniques: Expressing, Isolation, Derivative Assay

Journal: Life sciences

Article Title: Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

doi: 10.1016/j.lfs.2020.118974

Figure Lengend Snippet: Fig. 4. Intestinal SGLT3 protein is markedly upregulated in obese patients 6 months after Roux-en-Y gastric bypass (RYGB) surgery. (a) SGLT3 protein localize to jejunum epithelium of healthy and obese individuals. (b) qPCR and (c) Western blot analysis of jejunal biopsies from healthy volunteers and from obese patients before and 6 months after bariatric surgery. Data is normalized relative to mean of lean controls. Data was analysed by two-tailed paired t-test. **P < 0.01.

Article Snippet: Quantitative PCR was performed using HOT FIREPol Probe qPCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia) and respective assay (SLC5A4: Hs00429527_m1, LRP10: Hs01047362_m1, RPLP0: 4326314E, 18S rRNA: 4319413E; Thermo Fisher Scientific).

Techniques: Western Blot, Two Tailed Test

Journal: Nature neuroscience

Article Title: The medial preoptic area mediates depressive-like behaviors induced by ovarian hormone withdrawal through distinct GABAergic projections

doi: 10.1038/s41593-023-01397-2

Figure Lengend Snippet: a, RNAscope staining of Esr1, Vglut2 and Vgat in MPOA. Scale bar, 50um. b, Percentage of neurons in different specific categories. 80% of Esr1+ neurons were Vgat+. c, Dual-color CTb retrograde tracing of MPOA neurons projecting to VTA and PAG in Esr1-Cre::Ai14 mice. d, Images in MPOA. Arrows indicate Esr1+ (tdTomato+) neurons co-labelled with CTb647 (blue, from PAG) or CTb488 (green, from VTA). Scale bar, 100μm. e, Percentage of neurons in specific categories. 60% of Esr1+ neurons were labeled by either CTB647 or CTb488. Mean ± s.d., n = 3 animals. f, Average half-peak spike width of MPOA Vgat+ neurons for spikes evoked by current injections in NHW and HW slices. n=14 cells for NHW group and 12 cells for HW group, from 3 animals in each group. ****P < 0.0001, two-tailed Mann-Whitney test. g, Average trough voltage at different injection current amplitudes. n = 6, 6 cells for NHW and HW group, from 3 animals respectively. *P < 0.05, **P < 0.01, ***P < 0.001, two-way repeated measures ANOVA. h, Average half-peak spike width of spontaneous spikes of MPOA Esr1+ neurons in NHW (n=10 cells from 3 animals) and HW (n=10 cells from 3 animals) slices. ***P = 0.0002, two-tailed Mann-Whitney test. i, Average trough voltage of spontaneous spikes. n=10,10 cells for NHW and HW group, from 3 animals for each group. ***P = 0.0002, two-tailed Mann-Whitney test. For boxplot, centerline, mean, upper and lower end, 90 and 10 percentiles.

Article Snippet: Briefly, brain sections were hybridized with Esr1 (Advanced Cell Diagnostics, 478201), Vglut2 (Advanced Cell Diagnostics, 319171), Vgat (Advanced Cell Diagnostics, 319191) RNAscope probe mix, which were designed and validated by Adcanced Cell Diagostics.

Techniques: RNAscope, Staining, Retrograde Tracing, Labeling, Two Tailed Test, MANN-WHITNEY, Injection

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Progressive and pathway-wide disruption of cholesterol metabolism in mice with oligodendroglial TDP-43 deletion. (A) Overview of SREBF2 regulation on cholesterol biosynthesis and uptake (adapted from ). (B) Normalized gene expression data for Srebf2 and Srebf2-processing related genes. *, FDR-corrected P value <0.1 for Cnp -Cre + ; Tardbp fl/fl versus Cnp -Cre + ; Tardbp fl/+ contrast. Error bars describe expression SD, and significance was tested using the Wald test. Black bar: Tardbp fl/fl (control, ctrl); blue bar: Cnp -Cre; Tardbp fl/+ (conditional heterozygous knockout, cHet), and red bar: Cnp -Cre + ; Tardbp fl/fl (conditional knockout, cKO). n = 4 per genotypes for P21, n = 3 per genotypes for P60. (C) Overview of the cholesterol biosynthetic and uptake pathway. Srebf2 target track represents genes annotated as regulated by SREBF2. CLIP-seq track represents genes with CLIP-seq TDP-43 binding sites and location by combining our previous data and the public CLIP-seq assembly. +: Moderate confidence; two or more peaks on previous data or presence in two or more public datasets. ++: High confidence; more than six peaks on previous data or presence in four or more public datasets. Binding site location on gene in intronic region, exons, 5′-UTR, and 3′-UTR are colored green, blue, yellow, and orange, respectively. (D) Gene expression data of TDP-43 double-knockout samples reveal a near global down-regulation of cholesterol pathway–related genes. The phenotype was observed to progressively worse, with larger reductions in 60-d-old mice when compared with 21 d of age. *, FDR-corrected P value <0.1 for Cnp -Cre; Tardbp fl/fl versus Cnp -Cre; Tardbp fl/+ contrast. Error bars describe expression SD, and significance was tested using the Wald test. n = 4 per genotypes for P21, n = 3 per genotypes for P60. (E) Gene data summary for selected cholesterol-related genes containing gene structure model, CLIP-seq coverage (green), and RNA-seq coverage for Tardbp fl/fl (black, ctrl), Cnp -Cre; Tardbp fl/+ (blue, cHet), and Cnp -Cre; Tardbp fl/fl (red, cKO) conditions.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Disruption, Expressing, Knock-Out, Binding Assay, Double Knockout, RNA Sequencing Assay

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Reduced SREBF2 and its downstream targets in the TDP-43-deleted oligodendrocytes. (A) Confocal images of RNA-FISH of SREBF2, TDP-43, and LDLR assays using Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) conditions. Images shown are from dorsal gray, ventral gray, and white matter at P60. Scale bar: 20 µm. (B–D) Quantification of FISH signals across dorsal gray, ventral gray, and white matter for LDLR (B), SREBF2 (C), and TDP-43 (D). Puncta counts of individual oligodendrocytes (faded circle or triangle or square) and means (solid circle or triangle or square) for animal ( n = 3) plotted, total mean ± SEM derived from all animals. Significance was tested using unpaired t test; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n = 3 per genotype, at least three slices per animals were quantified. (E) Confocal images of RNA-FISH of TDP43 and IF of GSTP1 assays using Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) conditions. Images shown are from the white matter. Scale bar: 20 µm. (F) Quantification of FISH signals in white matter for TDP43 in GSTP1 + cells. Puncta counts of individual oligodendrocytes (faded circle or triangle) and means (solid circle or triangle) for each set of experiment ( n = 3) plotted, total mean ± SEM derived from all animals. Significance was tested using unpaired t test; ***, P < 0.001. n = 3 per genotype, at least three slices per animals were quantified. cKO, conditional knockout; ctrl, control.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Derivative Assay, Knock-Out

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Reduced SREBF2 and its downstream targets in the TDP-43–deleted oligodendrocytes. (A) LDLR and SREBF2 mRNA expression in oligodendrocytes of ctrl and cKO mouse lumbar spinal cord (arrowhead), revealed through combined RNA fluorescent in situ hybridization (RNA-FISH) and GST-P1 IF staining. Images were taken from the ventral gray matter at P21 and P60, at 20× magnification. Scale bar: 20 µm. 3D reconstruction of oligodendrocytes for LDLR (green) and SREBF2 (magenta) mRNA quantification. DAPI (blue). Scale bar: 3 µm. (B) Quantification of SREBF2 (i, ii) and LDLR (iii, iv) puncta in oligodendrocytes of ctrl and cKO mouse lumbar spinal cord, at P21 (i, iii) and P60 (ii, iv). Puncta counts for individual oligodendrocytes (faded circle and triangles), and means (solid circle and triangle) for each animal ( n = 3) plotted, total mean ± SEM derived from all animals. Significance was tested using unpaired t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 3 per genotype, 10 cells were quantified per section, and at least five sections were quantified per animal. (C) Confocal images of colabeling of oligodendrocytes (CC1+, red) and key proteins involved in cholesterol metabolism (SREBF2, left; HMGCS1, middle; and LDLR, right, green) in the white matter of spinal cord sections from 60-d-old mice. Square areas were separated into individual channels, indicating reduction of SREBF2, HMGCS1, and LDLR protein in oligodendrocytes. n = 3 per genotype, six to eight slices per animals were stained and observed. Scale bar: 10 µm. Enlarged images of single cell split into individual channels for SREBF2/HMGCS1/LDLR (green) and CC1 (red). Scale bar: 10 µm. cKO, conditional knockout; ctrl, control.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Expressing, In Situ Hybridization, Staining, Derivative Assay, Knock-Out

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: 3D reconstruction of SREBF2 and LDLR-FISH signals in oligodendrocyte. Representative oligodendrocyte from control mouse spinal cord reconstructed in 3D based on GST-P1 (red) IF signal and LDLR (green) and SREBF2 (purple) RNA-FISH signals.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques:

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Validation of SREBF2, LDLR, HMGCS1, and HMGCR antibodies. (A) Domain organization of human SREBF2 protein. Antibody epitope is within 455–469 aa. Cell lysates from (i) siRNA-control and siRNA-SREBF2, (ii) control plasmid and full-length cDNA expressing SREBF2. Black arrow: full-length SREBF2; blue arrow: N-terminal processed transcription domain. (B) Domain organization of human LDLR protein. Antibodies were produced using recombinant protein within 1–350 aa (for Proteintech, 10785–1-AP) or 500–550 aa (Novus, NBP1-06709-SS) for LDLR. Cell lysates from (i and iii) siRNA-control and siRNA-LDLR, and (ii and iv) control plasmid and full-length cDNA expressing GFP-LDLR. Black arrow: endogenous LDLR; green arrow: GFP-LDLR. (C) Domain organization of human HMGCS1 protein. Antibody was produced using recombinant protein within 171–520 aa. Cell lysates from (i) siRNA-control and siRNA-HMGCS1, and (ii) control plasmid and full-length cDNA expressing HMGCS1. Black arrow: endogenous HMGCS1. (D) Domain organization of human HMGCR protein. Cell lysates from (i) siRNA-control and siRNA-HMGCR, and (ii) control plasmid and full-length cDNA expressing HMGCR. Black arrow: endogenous HMGCR. ctrl, control.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Plasmid Preparation, Expressing, Produced, Recombinant

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Selective cholesterol reduction in the spinal cords of mice with oligodendroglial TDP-43 deletion. (A) Confocal images of astrocytes (GFAP, red) and microglia (Iba1, green) in of spinal cord sections from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice at P60. Scale bar: 200 µm. Enlarged images are from gray matter (middle) and white matter (right). Scale bar: 20 µm. n = 3 per genotype, approximately six to eight slices per animals were stained and observed. (B) Confocal images of astrocytes (GFAP, red) with LDLR (green) in the white matter of the spinal cord sections from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice at P60. Arrowhead indicates the colocalization of GFAP and LDLR signal in cKO mice. (C) Confocal images of astrocytes (Sox9, green) with SREBF2 (red) in the white matter of the spinal cord sections from Tardbp fl/fl (ctrl), and Cnp -Cre; Tardbp fl/fl (cKO) mice at P60. Arrowhead indicates the colocalization of Sox9 and SREBF2 signal in cKO mice. (D) Confocal images of microglia (CD45, green) with SREBF2 (red) in the white matter of the spinal cord sections from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice at P60. Arrowhead indicates the SREBF2 signals without CD45 in cKO mice. (E) Cholesterol level was measured in Tardbp fl/fl , Cnp -Cre; Tardbp fl/+ , and Cnp -Cre; Tardbp fl/fl spinal cord samples using GC/MS. The amount of cholesterol was normalized to protein content and expressed as micrograms cholesterol/milligrams protein. n = 5 per genotype per time point. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance; *, P < 0.05; ***, P < 0.001. (F) PC level was measured in Tardbp fl/fl , Cnp -Cre; Tardbp fl/+ , and Cnp -Cre; Tardbp fl/fl spinal cord samples using liquid chromatography–tandem MS. The amount of PC was normalized to protein content and compared with Tardbp fl/fl . n = 5 per genotype per time point. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance; *, P < 0.05. cKO, conditional knockout; ctrl, control.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Staining, Gas Chromatography-Mass Spectrometry, Liquid Chromatography, Knock-Out

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: TDP-43 regulates SREBF2 and LDLR expression in primary oligodendrocytes. (A) Schematic of primary oligodendrocyte culture using immunopanning from P7 spinal cords of Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice. (B) Confocal images of RNA-FISH of SREBF2, TDP-43, and LDLR assays using primary oligodendrocyte cultured from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice. (C and D) Quantification of FISH signals for SREBF2 (C) and LDLR (D) in ctrl and cKO oligodendrocytes. Puncta counts for individual oligodendrocytes (faded circle), and means (solid triangle) for each set of experiments ( n = 3) plotted, total mean ± SEM derived from all sets of experiments. Significance was tested using unpaired t test; **, P < 0.01. n = 3 per genotype, at least 20 cells were quantified per genotype per set of experiments. (E–H) Immunofluorescent staining of MBP (green; E and G), SREBF2 (red; E), LDLR (red; G), and TDP-43 (magenta; E and G) using differentiated oligodendrocytes from spinal cords of Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice, respectively. Oligodendrocytes without TDP-43 expression showed reduced SREBF2 (arrowhead) and LDLR (arrowhead). Scale bar = 10 µm. (F and H) Quantification of SREBF2 (F) and LDLR signal (H) in MBP-positive myelinating oligodendrocytes cultured from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice. Corrected total cell fluorescence of individual oligodendrocytes (faded circle), and means (solid triangle) for each set of experiment ( n = 3) plotted, total mean ± SEM derived from all sets of experiments. Significance was tested using unpaired t test; *, P < 0.05; ***, P < 0.001. n = 3 per genotype, at least 10 cells were quantified per genotype per set of experiment. (I) Immunoblots of LDLR, TDP-43, and MBP using lysates of primary oligodendrocytes cultured from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice. GAPDH was used as a loading control. cKO, conditional knockout; ctrl, control; spc, spinal cord; MW, molecular weight.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Expressing, Cell Culture, Derivative Assay, Staining, Fluorescence, Western Blot, Knock-Out, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: TDP-43 regulates cholesterol metabolism by regulating SREBF2 and LDLR expression. (A and B) TDP-43 binds to the SREBF2 transcripts as well as the downstream genes controlled by SREBF2. (A) Schematic of CLIP. Human oligodendrocytic (M03.13) cell lysates were UV cross-linked, lysed, and then used for TDP-43 pull-down assay. (B) Potential binding targets such as SERBF2 , HMGCR , HMGCS , LDLR , and positive control TARDBP transcripts were pulled down in the TDP-43 antiserum–treated samples. (C) Schematic of TDP-43 and SREBF2 RNAi experiments. (D) Acute knockdown of TDP-43 leads to down-regulation of (i) full-length and (ii) N-terminal SREBP2 and (iii) LDLR protein levels. Human oligodendrocyte cell lines (MO3.13) were transfected with control siRNA and siRNA against TDP-43 or SREBP2. Whole-cell lysates were probed with SREBP2, LDLR, and the loading control GAPDH. Quantifications of immunoblots for (i) full-length and (ii) N-terminal SREBP2 and (iii) LDLR protein levels. Quantifications were done with three independent batch of treatments ( n = 3) with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance, #, P < 0.10; *, P < 0.05; **, P < 0.01. (E) Newly synthesized transcripts detection by RT-qPCR in the MO3.13 cells treated with nontargeted siRNA or siRNA against TDP-43, followed by the DRB treatment. qPCR using primer pairs against the first exon-intron junction for (i) SREBF2 , (ii) LDLR, and (iii) RPLP0 detects the cDNAs from the cells collected in different intervals (0, 15, 30, and 60 min) after the removal of DRB. These gene expression levels were all normalized to no-treatment controls. Quantifications were done with three independent batch of treatments ( n = 3) with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance, ****, P < 0.0001. (F) Global translation reduction in the MO3.13 cells with TDP-43 knockdown. The lysates from cells treated with puromycin (100 µg/ml) in 15-min intervals (0, 15, and 30 min) after the incubation nontargeted siRNA or siRNA against TDP-43 were applied onto the SUnSET assay. Quantifications were done with two independent batches of treatments with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate statistical significance, ****, P < 0.0001. (G) The MO3 cells were treated with either control or TDP-43 siRNA followed by puromycin incubation. The cells were lysed followed by immunoprecipitation with an anti-puromycin antibody, and then were blotted with LDLR antibody. Ctrl, control; N-term, N-terminal; WB, Western blot; MW, molecular weight.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Expressing, Pull Down Assay, Binding Assay, Positive Control, Transfection, Western Blot, Synthesized, Quantitative RT-PCR, Incubation, Immunoprecipitation, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Characterization of mRNA levels and mRNA stability for SREBF2, HMGCR, and LDLR, and lipid droplets in MO3.13 cells with TDP-43 or SREBF2 knockdown. (A) RT-qPCR for (i) SREBF2, (ii) HMGCR, and (iii) LDLR in MO3.13 cells treated with siRNA-(negative) control, si-TDP-43, and si-SREBF2. The steady-state of SREBF2 and HMGCR mRNA was reduced in TDP-43 and SREBF2 knock-down conditions. Three independent experiments were performed, and three biological samples were performed per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance: *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (B) RNA half-lives for (i) SREBF2, (ii) HMGCR, and (iii) LDLR mRNAs in MO3.13 cells treated with siRNA-(negative) control and si-TDP-43. Three independent experiments were performed, and three biological samples were performed per experiment. (C) The relative cholesteryl-BODIPY fluorescent intensity in cells treated with control siRNA, siRNA against TDP-43, or SREBF2. *, P < 0.05; **, P < 0.01. Quantification was done from three independent experiments with >25 cells quantified per experiment. (D) Relative BODIPY-493/503 fluorescent intensity in cells treated with control siRNA, siRNA against TDP-43, or SREBF2. *, P < 0.05; **, P < 0.01. Quantification was done from three independent experiments with >40 cells quantified per experiment. (E) Relative Nile Red fluorescent intensity in cells treated with control siRNA, siRNA against TDP-43, or SREBF2. *, P < 0.05; **, P < 0.01. Quantification was done from three independent experiments with >25 cells quantified per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance for C–E.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Quantitative RT-PCR, Negative Control

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: SREBF2 and LDLR rescue cholesterol reduction caused by TDP-43 deletion. (A) MO3.13 cells were transfected with control siRNA and siRNA against TDP-43 or SREBF2. Cells were stained with TDP-43 (red), SREBF2 (green), and filipin (gray). Scale bar: 10 µm. (B) Relative filipin fluorescent intensity in cells treated with control siRNA and siRNA against TDP-43 or SREBF2. Quantification was done from three independent experiments with 90 cells quantified per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance; *, P < 0.05. (C) Total cholesterol level as measured by a fluorometric-based assay in cells treated with control siRNA and siRNA against TDP-43 or SREBF2. n = 3, one-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance; *, P < 0.05; **, P < 0.01. (D) Schematic of cDNA rescue experiment under TDP-43 knockdown conditions. (E) Immunoblotting for SREBF2 and LDLR in the rescue experiments. Blue arrow: full-length SREBF2; red arrow: transfected N-terminal domain of SREBF2; orange arrow: N-terminal domain of SREBF2; purple arrow: GFP-tagged LDLR; black arrow: endogenous LDLR. (F) Relative filipin fluorescent intensity in cells treated with control siRNA, or siRNA against TDP-43 with control rescue, or cDNA encoding SREBF2 or LDLR. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance; *, P < 0.05. Quantification was done from at least three independent experiments with 90 cells quantified per experiment. (G) Relative cholesterol level as measured by a fluorometric-based assay in cells treated with control siRNA, or siRNA against TDP-43 with control rescue, or cDNA encoding SREBF2 or LDLR. Three independent experiments were performed, and three biological samples were performed per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance; *, P < 0.05; **, P < 0.01.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Transfection, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination

doi: 10.1083/jcb.201910213

Figure Lengend Snippet: Cholesterol supplementation reverses myelin status in TDP-43–deleted oligodendrocytes. (A) Schematic of cholesterol supplementation experiments using primary oligodendrocytes cultured from Tardbp fl/fl (ctrl) and Cnp -Cre; Tardbp fl/fl (cKO) mice. Water-soluble cholesterol (β-cyclodextrin cholesterol complex) was incubated with cKO oligodendrocytes. (B) Confocal images of MBP and TDP-43 staining of oligodendrocytes from ctrl, cKO, and cKO with cholesterol supplementation (cKO + Chol). Scale bar is 20 µm. (C) Quantification of MBP area in oligodendrocytes from ctrl, cKO, and cKO with cholesterol supplementation (cKO + Chol). Cholesterol supplementation restores the myelination phenotype caused by TDP-43 deletion. MBP area of individual oligodendrocytes (faded circle), and means (solid triangle) for each set of experiment ( n = 3) plotted, total mean ± SEM derived from all sets of experiments. Significance was tested using unpaired t test, *, P < 0.05. Quantification was done from three independent experiments with at least 10 cells quantified per experiment. (D) Graphic summary and proposed model for how deletion of TDP-43 affects myelin via regulating SREBF2-mediated cholesterol metabolism in the oligodendrocytes. cKO, conditional knockout; ctrl, control.

Article Snippet: Sections were then washed twice in distilled water before incubation with RNAscope probe mixture (ACDbio; Mm-Ldlr-C1; cat #443701; or ACDbio; Mm-Srebf2-C3; cat #562261-C3; or ACDbio; Mm-Tardbp-C2; cat #518961-C2) at 40°C for 2 h. After probe incubation, slides were rinsed twice with 1× wash buffer (ACDBio; cat #310091) at RT for 2 min each.

Techniques: Cell Culture, Incubation, Staining, Derivative Assay, Knock-Out

Journal: iScience

Article Title: Allele-specific DNA demethylation editing leads to stable upregulation of allele-specific gene expression

doi: 10.1016/j.isci.2024.111007

Figure Lengend Snippet: Expression levels of the LY75 and FAM181B 6 days after locus or allele-specific DNA demethylation Values provided are ΔCq values normalized with the housekeeping gene SDHA. The negative controls NRT (sample subjected to cDNA conversion without the enzyme reverse transcriptase) and NTC (no template control) showed no detectable signal for the target regions and SDHA. The Cq values for SDHA were in the range of cycle 22–26 in all experiments. The experiments were conducted in three biological replicates (except for FAM181B-locus where only two replicates were obtained) and in each case, the Cq-value of sample was based on three technical replicates of the qPCR. The bars and errors report the average and standard deviation of the biological replicates. p values were determined by two-sided t test assuming equal variance. The untreated and scrambled sgRNA-EpiEditor treated sample collected on day 6 showed no detectable expression signal for the target genes. In this case, 40 cycles were used as limit of detection. n. s., not significant ( p value >0.05).

Article Snippet: ORATM SEE qPCR Probe Mix , highQu , QPP0401.

Techniques: Expressing, Reverse Transcription, Control, Standard Deviation