Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: TUG1 promotes AGE1.HN and PC12 cell death. (a) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells treated with and without hypoxia measured by western blot analysis normalized to β-actin (unpaired t test, ***p < 0.001. For c-caspase3: AGE1.HN: t(5.061) = −35.105, p < 0.001; PC12: t(8) = −25.322, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: t(8) = −49.07, p < 0.001; PC12: t(8) = −18.78, p < 0.001). Human or rat pcDNA-TUG1 and siRNA-TUG1 and their NCs were transfected into hypoxia-treated AGE1.HN and PC12 cells, respectively. (b) TUG1 expression in AGE1.HN and PC12 cells after transfection measured by RT-qPCR normalized to GAPDH (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 35,058.432, p < 0.001; PC12: F(3,16) = 70,046.402, p < 0.001). (c) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells after transfection measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.001. For c-caspase3: AGE1.HN: F(3,16) = 416.825, p < 0.001; PC12: F(3,16) = 260.449, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: F(3,16) = 475.2, p < 0.001; PC12: F(3,16) = 468.6, p < 0.001). (d) the death rate of AGE1.HN and PC12 cells after transfection measured by flow cytometry (one-way ANOVA, *p = 0.019, **p = 0.005, ***p < 0.001. AGE1.HN: F(3,16) = 184.670, p < 0.001; PC12: F(3,16) = 34.701, p < 0.001). (e) the distribution of TUG1 in AGE1.HN and PC12 cells evaluated by nuclear/cytoplasmic fractionation. Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Fractionation, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: BIK facilitates AGE1.HN and PC12 cell death. AGE1.HN and PC12 cells were delivered with pcDNA-BIK or siRNA-BIK. (a) the BIK mRNA expression in AGE1.HN and PC-12 cells determined by RT-qPCR (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 9423.939, p < 0.001; PC12: F(3,16) = 19,510.157, p < 0.001). (b) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells after transfection measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.01. For c-caspase3: AGE1.HN: F(3,16) = 750.932, p < 0.001; PC12: F(3,16) = 1644.487, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: F(3,16) = 583.2, p < 0.001; PC12: F(3,16) = 513.6, p < 0.001). (c) the death rate of AGE1.HN and PC12 cells after transfection measured by flow cytometry (one-way ANOVA, **p = 0.002, ***p < 0.001. AGE1.HN: F(3,16) = 648.677, p < 0.001; PC12: F(3,16) = 517.799, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Flow Cytometry, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: The regulatory role of TUG1/miR-338/BIK axis on rats with SCI (n = 5). (a) the BBB scores for hindlimb locomotion in the rats with ASCI injected with siRNA-TUG1 alone or with miR-338 inhibitor (one-way ANOVA, **p = 0.008, ***p < 0.001. F(3,16) = 175.553, p < 0.001). (b) detection of cell death in rat spinal cord by TUNEL staining (one-way ANOVA, ***p < 0.001. F(5,24) = 296.154, p < 0.001). (c) TUG1 (normalized to GAPDH) and miR-338 expression (normalized to U6) as well as the BIK mRNA expression (normalized to GAPDH) in spinal cord tissues of rats determined by RT-qPCR (one-way ANOVA, ***p < 0.001. TUG1: F(3,16) = 140.714, p < 0.001; miR-338: F(3,16) = 291.155, p < 0.001; BIK: F(3,16) = 251.190, p < 0.001). (d) the protein expression of c-caspase3 and c-caspase3/total caspase3 in spinal cord tissues of rats measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.001. For c-caspase3/total caspase3: F(3,16) = 146.102, p < 0.001. For c-caspase3/total caspase3: F(3,16) = 702.9, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Injection, TUNEL Assay, Staining, Expressing, Quantitative RT-PCR, Western Blot, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: Mechanisms of TUG1 mediated SCI progression. TUG1 interacts with miR-338, leading to BIK activation, which accelerates SCI via promoting cell death. While TUG1 knockdown downregulates BIK expression by interacting with miR-338, thus lowering c-caspase3 to hamper death

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Activation Assay, Expressing

Journal: bioRxiv

Article Title: CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation

doi: 10.1101/2024.10.19.619045

Figure Lengend Snippet: A - Illustration of modified transposase donor plasmids to test the effect of alternative NLSs on dCas9-KRAB localization and stability in neurons (drawn to scale). B - Flow cytometry plots measuring expression of modified dCas9-KRAB constructs or nCas9 via tagBFP fluorescence in neurons 14 days post induction of differentiation with dox from stably integrated iPSCs. Red line indicates the approximate cutoff for positive cells based on parental line autofluorescence. Plots represent a minimum of 3000 analyzed single cells. C - Quantification of flow cytometry data showing the mean fluorescence intensity of tagBFP normalized to autofluorescence of dCas9-KRAB constructs and nCas9 in Day 14 neurons (Parental autofluorescence=1). Alternative NLS sequences rescue dCas9-KRAB expression to levels comparable to nCas9, with the 2xMeCP2 NLS version having the highest expression with a mean population intensity of 4.931x the parental, compared to a mean of 4.586x for the MeCP2 NLS construct and 4.636x for the cMyc NLS+MeCP2 NLS construct. Data represent the mean fluorescence intensity of 2-3 wells per line with a minimum of 2000 analyzed single cells per sample. Error bars represent mean ± SD. D - Representative Western blots (left) and quantification (right) of total protein from Day 14 neurons probed for Cas9 showing significant rescue of total protein levels of 2xMeCP2 NLS-dCas9-KRAB compared to the original dCas9-KRAB, with levels comparable to nCas9. Cas9 antibody signal was normalized to TUBB3 levels. Data represents 3 independent wells per line. Error bars represent mean ± SD. Protein levels between lines were compared with One-way ANOVA with Tukey’s multiple comparisons test. ** P=0.0050 for dCas9-KRAB vs Cas9, * P=0.0150 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, ns P=0.5719 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. E-Representative Western blots (left) and quantifications (right) of dCas9-KRAB or nCas9 protein levels in nuclear and cytoplasmic protein fractions from Day 14 neurons. The original dCas9-KRAB and nCas9 are both predominantly cytoplasmic with nearly identical distributions despite much lower dCas9-KRAB levels. The 2xMeCP2 NLS-dCas9-KRAB construct shows a significant shift into the nuclear fraction which corresponds with the rescue of total protein levels. LaminB1 and GAPDH were used as nuclear and cytoplasmic controls, respectively, and were probed on replicate sample blots in parallel with Cas9. Quantifications for each protein represent the intensity of the signal in the respective fraction divided by the sum of intensities across both fractions. Cas9 quantifications were performed on respective LaminB1 and GAPDH blots. Data represent 3 independent wells per line. Error bars represent mean ± SD. Fractionation control and Cas9 antibody signals across cell lines were compared with 2-way ANOVA with Tukey’s multiple comparisons test. For Cas9 on LaminB1 blots: ns P=0.1236 for dCas9-KRAB vs nCas9, **** P<0.0001 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, **** P<0.0001 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. For Cas9 on GAPDH blots: ns P=0.6805 for dCas9-KRAB vs nCas9, **** P<0.0001 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, **** P<0.0001 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. F - Representative images of dCas9-KRAB protein localization in Day 14 neurons with ICC using a Cas9 antibody (green). Cells are stained with a MAP2 antibody (red) and nuclei with NucSpot 750/780 (magenta). Nuclei are outlined for reference. 2xMeCP2 NLS-dCas9-KRAB shows improved nuclear localization over original nCas9 and dCas9-KRAB constructs and higher intensity staining than the original dCas9-KRAB, consistent with the results from biochemistry assays. Images are Max IPs of 4-plane Z-stacks (0.6um) taken at 40x with a spinning disk confocal. All images are adjusted to the same LUTS, based on background Cas9 antibody staining in the Parental line. Scale bars represent 20um. G - Quantification of RT-qPCR data showing significantly improved knockdown of PSAP (top) and SNCA (bottom) in neurons expressing the 2xMeCP2 NLS-dCas9-KRAB vs the original dCas9-KRAB. Neurons were transduced with lentivirus expressing an mScarlet marker with either a targeting or non-targeting sgRNA at Day 14 (PSAP) or Day 21 (SNCA) post-differentiation and RNA harvested 7-days post-transduction. Gene expression levels were normalized to GAPDH and ACTB and are shown relative to the non-targeting average. Data represent 3 independent wells per transduction per line. 2-way ANOVA with uncorrected Fisher’s LSD was used to compare targeting guide with non-targeting guide within cell lines and targeting guides across cell lines. For PSAP KDs: ns P=0.0745 for dCas9-KRAB+NTCg vs +PSAPg, *** P=0.0004 for 2xMeCP2 NLS-dCas9-KRAB+NTCg vs +PSAPg, ** P=0.006 for dCas9-KRAB+PSAPg vs 2xMeCP2 NLS-dCas9-KRAB+PSAPg. For SNCA KDs: * P=0.0152 for dCas9-KRAB+NTCg vs +SNCAg, **** P<0.0001 for 2xMeCP2 NLS-dCas9-KRAB+NTCg vs +SNCAg, ** P=0.0035 for dCas9-KRAB+SNCAg vs 2xMeCP2 NLS-dCas9-KRAB+SNCAg.

Article Snippet: The following primary antibodies were used: Cas9 (Cell Signaling, 14697; 1:1000), TUBB3 (Abcam, ab18207; 1:800), LaminB1 (Proteintech, 12987-1-AP; 1:2000), GAPDH (Cell Signaling, 2118; 1:500).

Techniques: Modification, Flow Cytometry, Expressing, Construct, Fluorescence, Stable Transfection, Western Blot, Fractionation, Control, Staining, Quantitative RT-PCR, Knockdown, Transduction, Marker