ctcf shrna lentiviral particles  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased TREK1 expression upon Aβo treatment, which is markedly reduced by co-treatment with the calcium chelator BAPTA-AM. b, Quantification of TREK1 fluorescence intensity following Aβ42o and/or BAPTA-AM treatment (n=61-90 Cells; **p < 0.01, ####p < 0.0001; one-way ANOVA with Šidák’s test). c, Representative images showing that TREK1 increase induced by Aβ42o is attenuated by the AC1 inhibitor ST034307. d, Quantification of TREK1 fluorescence intensity following Aβ42o and/or ST034307 treatment (n=23-31 cells; **p < 0.01, ###p < 0.001; one-way ANOVA with Šidák’s test). e, Representative images showing TREK1 expression is decreased in Aβ42o treated neurons transfected with AC1-specific siRNA but not with scrambled (Sc) siRNA. f, Quantification of TREK1 fluorescence intensity following Aβ42o treatment with AC1 knockdown (n=71-128 cells; ****p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). g, Representative images showing TREK1 expression is reduced in Aβ42o-treated neurons transfected with AC8-specific siRNA, but not with scrambled siRNA. h, Quantification of TREK1 fluorescence intensity following Aβ42o treatment with AC8 knockdown (n=14-19 cells; ****p < 0.0001, unpaired t-test). i, Treatment with the cAMP analog 8-CPT-cAMP increases TREK1 expression in primary neurons. j, Quantification of TREK1 fluorescence intensity following 8-CPT- cAMP treatment (n=57-62 cells; ***p < 0.001, unpaired t-test). k, Treatment with forskolin, a cAMP activator, mimics Aβ42o by increasing TREK1 expression in primary neurons. l, Quantification of TREK1 fluorescence intensity following forskolin treatment (n=146-242 cells; ****p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). m, Schematic representation illustrating that Aβ42o-induced TREK1 upregulation is mediated by calcium influx via the AC1/AC8–cAMP signaling pathway. Data are presented as mean ± SEM. 3-5 independent cultures per group were used.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Immunofluorescence, Expressing, Fluorescence, Transfection, Knockdown

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased TREK1 expression in Aβ42o-treated neurons, which is decreased upon co-treatment with the PKA inhibitor KT5720. b, Quantification of TREK1 fluorescence intensity following Aβ42o and/or KT5720 treatment (n = 52-70 cells; ****p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). c, Representative images showing that TREK1 increase induced by Aβ42o is attenuated by another PKA inhibitor H89. d, Quantification of TREK1 fluorescence intensity following Aβ42o and/or H89 treatment (n = 43–49 cells; **** p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). e, Schematic representation of the TREK1 locus on chromosome 1q41 showing predicted promoters (blue boxes; P1–P3) and ENCODE-annotated CTCF binding sites (red boxes; accession IDs indicated). Genomic positions are indicated relative to the transcription start site (TSS). ReMap ChIP-seq and density tracks demonstrate experimental support for the predicted binding sites, with peaks at site E1421793 located proximal to the promoter (P1). Motif analysis using JASPAR confirmed the presence of a consensus CTCF motif within this region (right panel), with associated FIMO statistics (score, p-value, and q-value). The identified site (highlighted in red) spans chr1:215076354–215076685 (band 1q41), has a genomic size of 332 bp, and is classified as “CTCF-bound” in ENCODE. f, Representative images showing decreased TREK1 expression in Aβ42o treated neurons transfected with CTCF-specific siRNA compared to scrambled (Sc) siRNA. g, Quantification of TREK1 fluorescence intensity following Aβ42o and/or CTCF knockdown (n = 37–76 cells; **** p < 0.0001, #### p < 0.0001; one-way ANOVA with Šidák’s test). h, Representative images showing decreased TREK1 expression in hippocampal neurons of 3xTg mice following intrahippocampal injection of CTCF shRNA lentivirus compared to control. i, Quantification of TREK1 fluorescence intensity in 3xTg mice after CTCF knockdown (n = 13–19 sections; ****p < 0.0001; unpaired t-test). j, Schematic representation of the PKA–CTCF signaling axis in regulating Aβ42- induced TREK1 expression. Data are presented as mean ± SEM. 3-5 independent cultures or animals per group were used.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Immunofluorescence, Expressing, Fluorescence, Binding Assay, ChIP-sequencing, Transfection, Knockdown, Injection, shRNA, Control

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative calcium imaging traces from control and Aβ42o treated neurons showing that Aβ42o increases spontaneous calcium transient frequency. This hyperexcitability is further enhanced by the TREK1 inhibitor spadin and suppressed by the TREK1 activator BL-1249. b, Quantification of calcium event frequency upon treatment with Aβ42o/spadin/BL-1249 (n = 512–1002 cells; ****p < 0.0001, ##p < 0.01, †p < 0.05; one-way ANOVA with Šidák’s test). c, Representative FluoVolt traces measuring membrane potential fluctuations manifest enhanced neuronal activity in the presence of TREK1 inhibitor spadin and suppressed neuronal activity by the TREK1 activator BL-1249 compared to Aβ42o treatment alone. d, Quantification of potential spike frequency upon treatment with Aβ42o/spadin/BL- 1249 (n = 31–105 cells; **p < 0.01, #p < 0.05, ††p < 0.01; one-way ANOVA with Šidák’s test). e, Representative calcium traces from neurons treated with Aβ42o along with scrambled (Sc) siRNA, KCNK2 siRNA, or a KCNK2 overexpression (OE) construct. f, Quantification of calcium event frequency upon knocking down KCNK2 in the presence of Aβ42o treatment (n = 49–67 cells; *p < 0.05; unpaired t-test). g, Quantification of calcium event frequency upon overexpressing KCNK2 in the presence of Aβ42o treatment (n = 37–48 cells; *p < 0.05, #p < 0.05; one-way ANOVA with Šidák’s test). h, Representative patch-clamp recordings of action potentials in control, Aβ42o, and Aβ42o + spadin treated neurons showing exacerbated action potential firing with TREK1 blockade. i, Quantification of action potential frequency following Aβ42o and/or spadin treatment (n = 17 cells; *p < 0.05, #p < 0.05; one-way ANOVA with Šidák’s test). j, Resting membrane potential (RMP) is more depolarized in Aβ42o treated neurons compared to control, and further depolarizes in presence of spadin with Aβ42o (n = 19 cells; ****p < 0.0001, #p < 0.05; one-way ANOVA with Šidák’s test). k, Representative traces showing excitatory postsynaptic current (EPSC) frequency is increased in neurons treated with Aβ42o + spadin compared to Aβ42o alone. l, Quantification of EPSC frequency (n = 19 cells; *p < 0.05, ##p < 0.01; one-way ANOVA with Šidák’s test). m, Quantification of EPSC amplitude (n = 19 cells). n, Representative traces showing Inhibitory postsynaptic current (IPSC) frequency is decreased in neurons treated with Aβ42o+spadin compared to Aβ42o alone. o, Quantification of IPSC frequency (n = 10 cells; **p < 0.01, #p < 0.05; one-way ANOVA with Šidák’s test). p, Quantification of IPSC amplitude (n = 10 cells). q, Representative ex vivo calcium imaging heat map from hippocampal slices of 3xTg mice injected with TREK1 shRNA lentivirus showing elevated calcium activity compared to sc shRNA-injected mice. r, Representative calcium imaging traces demonstrating increased calcium transient frequency following TREK1 knockdown. s, Quantification of calcium event frequency in TREK1 knockdown mice compared to sc shRNA-injected mice (n = 13–27 cells; *p < 0.05; unpaired t-test). Data are presented as mean ± SEM from 3-5 independent cultures.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Imaging, Control, Membrane, Activity Assay, Over Expression, Construct, Patch Clamp, Ex Vivo, Injection, shRNA, Knockdown

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased VGLUT1 intensity in the hippocampus of 3xTg mice 15 days after intrahippocampal injection with TREK1 shRNA lentivirus compared with vehicle-injected controls. b, Representative images showing decreased VGAT intensity under the same TREK1 knockdown conditions. c, Quantification of VGLUT1 fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 67–76 sections; ****p < 0.0001; unpaired t-test). d, Quantification of VGAT fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 60–63 sections; ****p < 0.0001; unpaired t-test). e, Quantification of Excitatory/inhibitory (E/I) ratio, calculated as VGLUT1/VGAT intensity, is markedly elevated in TREK1 knockdown mice compared with vehicle injected controls (n = 3– 4; **p < 0.01; unpaired t-test). f, Representative immunofluorescence images demonstrating enhanced Aβ deposition in the hippocampus of TREK1 knockdown 3xTg mice compared with vehicle-injected controls. g, Quantification of Aβ fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 50 sections; *p < 0.05; unpaired t-test). h, Quantification showing a significant decrease in MAP2 intensity in the hippocampus of 3xTg mice injected with TREK1 shRNA compared with scrambled (Sc) shRNA controls (n = 159–166 sections; ****p < 0.0001; unpaired t-test). i, Schematic representation illustrating the effects of TREK1 knockdown on excitatory/inhibitory balance. Data are expressed as mean ± SEM from 3–4 mice per group.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Immunofluorescence, Injection, shRNA, Knockdown, Fluorescence

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: The diagram summarizes the signaling cascade by which Aβ42 induced neuronal hyperexcitability drives TREK1 upregulation via calcium influx and the AC1/AC8–cAMP–PKA–CTCF axis. TREK1 upregulation decreases neuronal excitability, limits excitatory/inhibitory balance, thereby improves neuronal health in 3xTg mice.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques:

Journal: Cell Research

Article Title: Tau is a receptor with low affinity for glucocorticoids and is required for glucocorticoid-induced bone loss

doi: 10.1038/s41422-024-01016-0

Figure Lengend Snippet: a Immunoblotting of pTau S422, S396 and S202/T205 in Raw264.7 cells treated with low (L, 10 nM) or high (H, 10 µM) dose of dexamethasone along with 50 ng/mL RANKL for the indicated time. GAPDH is used as a loading control. Representative images from duplicate results are shown. b Immunoblotting of Tau in Tau knockout Raw264.7 cells transfected with empty vector, full length (FL) or Tau with serial point mutations. Representative images of duplicate results are shown. c , d Representative bright-field images ( c ) and corresponding quantification ( d ) of TRAP-positive multinuclear osteoclasts differentiated from FL- or Tau with serial point mutations- transfected Tau −/− Raw264.7 macrophage treated with 50 ng/mL RANKL and 10 µM dexamethasone for 5 days. Scale bar = 100 µm. e Confocal images of Raw264.7 cells labeled with antibodies for Tau and tubulin (left panel), and corresponding fluorescence signal intensity plots of Tau (red) and tubulin (green) vesicles (right panel). Cells are treated with or without 50 ng/mL RANKL and 10 µM dexamethasone. Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. f Confocal images of Raw264.7 cells labeled with antibodies for p-Tau Ser422 and tubulin (left panel), and corresponding fluorescence signal intensity plots of p-Tau Ser422 (red) and tubulin (green) vesicles (right panel). Cells are treated with or without 50 ng/mL RANKL and 10 µM dexamethasone. Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. g Experimental design and principle used to identify the potential kinases that are involved in dexamethasone-dependent p-Tau Ser422. h Summary of the potential kinases responsible for dexamethasone-dependent activation of Tau. i Knockdown efficiency of TTBK1 in Raw264.7 cells, measured by western blot ( n = 3). j Immunoblotting of dexamethasone-activated p-Tau Ser422 in Raw264.7 cells transfected with TTBK1 siRNA or treated with different kinase inhibitors, as indicated. k Densitometry analysis of immunoblotting results shown in j . l , m Representative bright-field images ( l ) and corresponding quantification ( m ) of TRAP-positive multinuclear osteoclast differentiated from Raw264.7 macrophages, which are transfected with TTBK1 siRNA or treated with different kinase inhibitor, followed by stimulating with 50 ng/mL RANKL and 10 µM dexamethasone for 5 days. Scale bar = 100 µm. n Confocal images of vehicle- or 10 µM dexamethasone-treated Raw264.7 cells labeled with antibodies for Tau and TTBK1 (left panel), and corresponding fluorescence signal intensity plots of Tau (red) and TTBK1 (green) vesicles (right panel). Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. o IP from 10 µM dexamethasone-treated Raw264.7 cells with anti-Tau antibody, and detection of TTBK1 by immunoblotting. Representative image of duplicate results is shown. p IP from 10 µM dexamethasone-treated Raw264.7 cells transfected with FLAG-tagged FL Tau or Tau with serial deletion mutations using anti-FLAG M2 resins, and detection of TTBK1 by immunoblotting ( n = 3). Data are means ± SD. P values are calculated by one-way ANOVA with Bonferroni post-hoc test ( d , k , m ).

Article Snippet: To knock down TTBK1 and CTCF in Raw264.7 cells, cells were transfected with TTBK1 siRNA (sc-154747, Santa Cruz), CTCF siRNA (SR419676, Origene) or scramble negative control siRNA (SR30004, Origene) using Lipofectamine 2000 (11668019, Invitrogen) for 48 h. After transfection, the knockdown efficiency was determined by western blot.

Techniques: Western Blot, Control, Knock-Out, Transfection, Plasmid Preparation, Labeling, Fluorescence, Activation Assay, Knockdown

Journal: Cell Research

Article Title: Tau is a receptor with low affinity for glucocorticoids and is required for glucocorticoid-induced bone loss

doi: 10.1038/s41422-024-01016-0

Figure Lengend Snippet: a Schematic for application of biochemical co-purification and mass spectrometry approaches to screen the transcriptional factors recruited to activated Tau by high-dose dexamethasone. Raw264.7 cells transfected with FLAG or FLAG-tagged Tau are treated with or without 10 µM dexamethasone for 30 min, followed by precipitation with anti-FLAG M2 resins. b Summary of the identified transcriptional factors recruited to Tau in 10 µM dexamethasone-treated Raw264.7 cells. c Knockdown efficiency of CTCF in Raw264.7 cells, measured by immunoblotting. d , e Representative bright-field images of TRAP-positive multinuclear osteoclast differentiated from Raw264.7 macrophages treated with 50 ng/mL RANKL and 10 µM dexamethasone for 5 days. Raw264.7 cells are treated with andrographolide (AGL, p50 inhibitor) or transfected with CTCF siRNA before differentiating into osteoclasts. Scale bar = 100 µm. f – h Gene expression levels of osteoclast differentiation markers NFATc1 (following 3 days of differentiation), CTSK and CTR (following 5 days of differentiation) determined by qRT-PCR. Bone marrow-derived macrophage isolated from WT, Tau −/− and GR −/− mice are differentiated with 20 ng/mL M-CSF and 50 ng/mL RANKL supplemented with low or high dose of dexamethasone ( n = 3). i IP from 10 µM dexamethasone-treated Raw264.7 cells with anti-Tau antibody, and detection of p105/p50 by immunoblotting. Representative image of duplicate results is shown. j IP from 10 µM dexamethasone-treated Raw264.7 cells transfected with FLAG-tagged FL or serial deletion mutations of Tau with anti-FLAG M2 resins, and detection of p105/p50 by immunoblotting. k , l Immunoblotting analysis ( k ) and quantification ( l ) of relative levels of p105 and p50 in control, Tau −/− and GR −/− Raw264.7 cells after 10 µM dexamethasone stimulation ( n = 3). m , n Immunoblotting analysis ( m ) and quantification ( n ) of relative levels of p105 and p50 in TTBK1 siRNA-transfected Raw264.7 cells after 10 µM dexamethasone stimulation. o , p Confocal images of Raw264.7 cells labeled with antibodies for p105/p50 ( o ), and relative fluorescence intensity of p105/p50 in cytoplasm and nuclear are shown ( p ) ( n = 3 biological replicates). q , r ChIP-qPCR assay of p50 in the NFATc1 promoter in WT ( q ) or GR −/− ( r ) Raw264.7 cells treated with 50 ng/mL RANKL for 1 h. s ChIP-qPCR assay of p50 in the NFATc1 promoter in Tau −/− Raw264.7 cells transfected with or without FL Tau or Tau S422P mutant followed by treatment with 50 ng/mL RANKL for 1 h. t , u ChIP-qPCR assay of p50 ( t ) and p65 ( u ) in the NFATc1 promoter in control or p50 siRNA knockdown Raw264.7 cell treated with RANKL in the presence or absence of Trx0237 for 1 h. Data are means ± SD. P values are calculated by one-way ANOVA with Bonferroni post-hoc test ( f – h , n , s – u ), two-way ANOVA with Bonferroni post-hoc test ( p ) and two-tailed unpaired Student’s t -test ( l , q , r ). n.s., not significant; ** P < 0.01.

Article Snippet: To knock down TTBK1 and CTCF in Raw264.7 cells, cells were transfected with TTBK1 siRNA (sc-154747, Santa Cruz), CTCF siRNA (SR419676, Origene) or scramble negative control siRNA (SR30004, Origene) using Lipofectamine 2000 (11668019, Invitrogen) for 48 h. After transfection, the knockdown efficiency was determined by western blot.

Techniques: Copurification, Mass Spectrometry, Transfection, Knockdown, Western Blot, Gene Expression, Quantitative RT-PCR, Derivative Assay, Isolation, Control, Labeling, Fluorescence, ChIP-qPCR, Mutagenesis, Two Tailed Test