Journal: bioRxiv

Article Title: Cancer-associated KBTBD4 mutations induce differentiation defects and confer a unique therapeutic vulnerability

doi: 10.64898/2026.03.12.711277

Figure Lengend Snippet: (A) Volcano plot showing global protein expression in HL60 cells transduced with P311PP mutant. Blue and red dots show significantly decreased or increased proteins (adjusted p-value<0.05). CoREST components RREB1, RCOR1, KDM1A, RCOR3, and ZNF217 are highlighted in the volcano plot. The global proteome data was plotted after excluding PCDHGA11. (B) High throughput compound screening in HL60 cells stably expressing RCOR1-GFP treated with UM171 (200nM). Schematic of the screening rationale (top panel). The compounds were added at a final concentration of 1µM, and flow analysis was performed after 3 hours post treatment to measure the relative GFP expression (bottom panel). Red dots show the hits from the screen and the table on right-side shows the functional classification of the hits. (C) A dose-titration experiment showing the rescue of RCOR1-GFP by two class I specific HDAC inhibitors (mocetinostat and romidepsin) and three Pan HDAC inhibitors (belinostat, pracinostat and vorinostat). GFP mean fluorescence intensity of DMSO treatment was used as controls. Data from 3 replicates from 1 of 2 independent experiments with similar results are shown. (D) RCOR1 protein levels in P311PP expressing HL60 cells either treated with DMSO, belinostat (320nM) or mocetinostat (300nM) for 3 hours. (E) RCOR1 ELM2-GFP clones were expressed in HL60 cells and analyzed for the interaction with HDAC2 through immunoprecipitation. The degradation profiles of the corresponding alanine substitution clones to UM171 treatment are represented as a heat map. (F) Schematic representation of the mechanistic basis of HDAC inhibitors in preventing the CoREST degradation.

Article Snippet: The proteins were transferred to iBlot 2 PVDF membranes (#IB24001, Thermo Fisher Scientific) and probed with the following primary antibodies: RCOR1 (#14567), KDM1A (LSD1, #2184S), HDAC2 (#5113T, all from Cell Signaling Technology), GFP (#ab290, Abcam), and Actin (#612656, BD Biosciences).

Techniques: Expressing, Transduction, Mutagenesis, High Throughput Screening Assay, Stable Transfection, Concentration Assay, Functional Assay, Titration, Fluorescence, Clone Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Identification of the involvement of HDAC2 in bone metastasis-induced pain . A – D , RT-qPCR analysis of Hdac1 ( A ), Hdac3 ( B ), Hdac8 ( C ), and Hdac2 ( D ) abundance in L4/5 DRG tissues from sham and bone metastasis model rats at 14 days after surgery. n = 7 rats per group. E and F , western blotting analysis of HDAC2 abundance in L4/5 DRG tissues from sham and bone metastasis model rats at 14 days after surgery. n = 6 rats per group. G and H , chromatin immunoprecipitation assays of HDAC2 binding to Clc-3 gene promoter in ipsilateral L4/5 DRG tissues from sham and bone metastasis model rats at 14 days after surgery. G , representative ChIP assay result. H , summary of quantitative PCR quantification analysis. n = 8 rats per group. I – K , colocalization of HDAC2 with ClC-3 in DRG tissues. I , representative images showing the colocalization of HDAC2 with ClC-3 in ipsilateral L4/5 DRG tissues from bone metastasis model rats and sham controls at 14 days after surgery. The scale bar represents 50 μm. J and K , violin plot shows the mean fluorescence intensity of HDAC2 ( J ) and ClC-3 ( K ) in DRG tissues from bone metastasis model rats and sham controls at 14 days after surgery. (n = 54–61 cells from three rats per group). Data are presented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( K ). ChIP, chromatin immunoprecipitation; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Fluorescence, Histone Deacetylase Assay

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Hdac2 in DRG neurons increases the expression of ClC-3 channels, reduces the neuronal excitability and attenuates pain hypersensitivity in bone metastasis model rats . A , western blotting analysis of HDAC2 protein abundance in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shHDAC2 treated rats at 14 days after tumor cells inoculation. n = 6 rats per group. B , chromatin immunoprecipitation assays of HDAC2 binding to Clc-3 gene promoter in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shHDAC2 treated rats at 14 days after tumor cells inoculation. n = 8 rats per group. C and D , RT-qPCR analysis and western blotting analysis of ClC-3 mRNA and protein abundance in L4/5 DRG tissues from intrathecal LV-GFP and LV-shHDAC2 treated rats at 14 days after tumor cells inoculation. n = 4 to 6 rats per group. E – G , electrophysiological analyses of neuronal excitability in ipsilateral L4/5 DRG neurons of bone metastasis model rats that received intrathecal LV-GFP and LV-shHDAC2, recorded at 14 days after tumor cells inoculation. E , representative neuronal action potentials evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. The scale bar represents 20 mV, 100 ms. F and G , plots of threshold potential and rheobase are shown. n = 10∼20 cells from 3 rats per group. H and I , assessment of ipsilateral PWT ( H ) and PWL ( I ) from intrathecal LV-GFP and LV-shHDAC2 treated rats after tumor cells inoculation. n = 10 rats per group. J , assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration. n = 10 rats per group. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, unpaired t test for ( A )–( G ); two-way ANOVA with Sidak’s post hoc test for ( H )–( J ). DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; LV-GFP, lentivirus expressing green fluorescent protein; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing, Western Blot, Quantitative Proteomics, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Contribution of IGF1 to the activation of AKT signaling and HDAC2-mediated Clc-3 transcription repression in DRG neurons, and induces pain hypersensitivity in na39;ve rats . A and B , RT-qPCR analysis and western blotting analysis of IGF1 mRNA and protein abundance in L4/5 DRG tissues from sham and BCP rats at 14 days after tumor cells inoculation. n = 4 to 8 rats per group. C – E , western blotting analysis of phosphorylated AKT (p-AKT, D ) and AKT ( E ) protein abundance in ipsilateral L4/5 DRG tissues obtained from vehicle and IGF1-treated naïve rats. n = 6 rats per group. C , representative blots are shown. F and G , western blotting analysis of HDAC2 protein abundance in ipsilateral L4/5 DRG tissues obtained from vehicle and IGF1-treated naïve rats. n = 6 rats per group. H – J , RT-qPCR analysis and western blotting analysis of ClC-3 mRNA and protein abundance in L4/5 DRG tissues from vehicle and IGF1-treated naïve rats. n = 4 to 8 rats per group. K and L , assessment of the PWT ( K ) and PWL ( L ) for naïve rats that received intrathecal IGF1 or vehicle, performed at 5 days after drug administration (n = 10 rats per group). M , assessment of animal’s locomotor function before and after intrathecal drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( J ); two-way ANOVA with Sidak’s post hoc test for ( K )–( M ). BCP, bone cancer pain; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; IGF1, insulin-like growth factor 1; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Igf1r in DRG neurons disrupts intrathecal IGF1-induced activation of AKT signaling as well as the upregulated expression of HDAC2, the decreased expression of ClC-3 protein and pain hypersensitivity in na9;ve rats . A and B , RT-qPCR analysis and western blotting analysis of IGF1R mRNA and protein abundance in L4/5 DRG tissues from sham and BCP rats at 14 days after tumor cells inoculation. n = 6–7 rats per group. C , representative images showing the colocalization of IGF1R, HDAC2, and ClC-3 in DRG neurons. The scale bar represents 25 μm. D – F , western blotting analysis of p-AKT ( E ) and AKT ( F ) protein abundance in ipsilateral L4/5 DRG tissues obtained from IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP, respectively (n = 4 rats per group). D , representative blots are shown. G – I , western blotting analysis of HDAC2 ( H ) and ClC-3 ( I ) protein abundance in ipsilateral L4/5 DRG tissues obtained from IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP, respectively (n = 4 rats per group). G , representative blots are shown. J and K , assessment of ipsilateral PWT and PWL for IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP (n = 10 rats per group). L , assessment of animal’s locomotor function before and after intrathecal drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( B ); one-way ANOVA followed by Dunnett's post hoc test for ( E )–( I ) two-way ANOVA with Sidak’s post hoc test for ( J )–( L ). BCP, bone cancer pain; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; IGF1, insulin-like growth factor 1; LV-GFP, lentivirus expressing green fluorescent protein; p-AKT, phosphorylated AKT; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Igf1r in DRG neurons impairs HDAC2-mediated transcriptional repression of Clc-3 gene, reduces neuronal excitability and attenuates pain hypersensitivity in bone metastasis model rats . A – E , western blotting analysis of p-AKT ( B ), AKT ( C ), HDAC2 ( D ) and ClC-3 ( E ) abundance in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shIGF1R treated rats at 14 days after tumor cells inoculation. n = 4 to 6 rats per group. A , representative blots are shown. F – H , electrophysiological analyses of neuronal excitability in ipsilateral L4/5 DRG neurons of bone metastasis model rats that received intrathecal LV-GFP and LV-shIGF1R, recorded at 14 days after tumor cells inoculation. F , representative neuronal action potentials evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. The scale bar represents 20 mV, 100 ms. G and H , plots of threshold potential and rheobase are shown. n = 10∼20 cells from three rats per group. I and J , assessment of ipsilateral PWT ( I ) and PWL ( J ) from intrathecal LV-GFP and LV-shIGF1R treated rats after tumor cells inoculation. n = 10 rats per group. K , assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration. n = 10 rats per group. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( B )–( H ); two-way ANOVA with Sidak’s post hoc test for ( I )–( K ). DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; LV-GFP, lentivirus expressing green fluorescent protein; p-AKT, phosphorylated AKT; PWL, paw withdrawal latency; PWT, paw withdrawal threshold.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Histone Deacetylase Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Identification of the involvement of HDAC2 in bone metastasis-induced pain . A – D , RT-qPCR analysis of Hdac1 ( A ), Hdac3 ( B ), Hdac8 ( C ), and Hdac2 ( D ) abundance in L4/5 DRG tissues from sham and bone metastasis model rats at 14 days after surgery. n = 7 rats per group. E and F , western blotting analysis of HDAC2 abundance in L4/5 DRG tissues from sham and bone metastasis model rats at 14 days after surgery. n = 6 rats per group. G and H , chromatin immunoprecipitation assays of HDAC2 binding to Clc-3 gene promoter in ipsilateral L4/5 DRG tissues from sham and bone metastasis model rats at 14 days after surgery. G , representative ChIP assay result. H , summary of quantitative PCR quantification analysis. n = 8 rats per group. I – K , colocalization of HDAC2 with ClC-3 in DRG tissues. I , representative images showing the colocalization of HDAC2 with ClC-3 in ipsilateral L4/5 DRG tissues from bone metastasis model rats and sham controls at 14 days after surgery. The scale bar represents 50 μm. J and K , violin plot shows the mean fluorescence intensity of HDAC2 ( J ) and ClC-3 ( K ) in DRG tissues from bone metastasis model rats and sham controls at 14 days after surgery. (n = 54–61 cells from three rats per group). Data are presented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( K ). ChIP, chromatin immunoprecipitation; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Fluorescence, Histone Deacetylase Assay

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Hdac2 in DRG neurons increases the expression of ClC-3 channels, reduces the neuronal excitability and attenuates pain hypersensitivity in bone metastasis model rats . A , western blotting analysis of HDAC2 protein abundance in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shHDAC2 treated rats at 14 days after tumor cells inoculation. n = 6 rats per group. B , chromatin immunoprecipitation assays of HDAC2 binding to Clc-3 gene promoter in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shHDAC2 treated rats at 14 days after tumor cells inoculation. n = 8 rats per group. C and D , RT-qPCR analysis and western blotting analysis of ClC-3 mRNA and protein abundance in L4/5 DRG tissues from intrathecal LV-GFP and LV-shHDAC2 treated rats at 14 days after tumor cells inoculation. n = 4 to 6 rats per group. E – G , electrophysiological analyses of neuronal excitability in ipsilateral L4/5 DRG neurons of bone metastasis model rats that received intrathecal LV-GFP and LV-shHDAC2, recorded at 14 days after tumor cells inoculation. E , representative neuronal action potentials evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. The scale bar represents 20 mV, 100 ms. F and G , plots of threshold potential and rheobase are shown. n = 10∼20 cells from 3 rats per group. H and I , assessment of ipsilateral PWT ( H ) and PWL ( I ) from intrathecal LV-GFP and LV-shHDAC2 treated rats after tumor cells inoculation. n = 10 rats per group. J , assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration. n = 10 rats per group. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, unpaired t test for ( A )–( G ); two-way ANOVA with Sidak’s post hoc test for ( H )–( J ). DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; LV-GFP, lentivirus expressing green fluorescent protein; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing, Western Blot, Quantitative Proteomics, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Contribution of IGF1 to the activation of AKT signaling and HDAC2-mediated Clc-3 transcription repression in DRG neurons, and induces pain hypersensitivity in na39;ve rats . A and B , RT-qPCR analysis and western blotting analysis of IGF1 mRNA and protein abundance in L4/5 DRG tissues from sham and BCP rats at 14 days after tumor cells inoculation. n = 4 to 8 rats per group. C – E , western blotting analysis of phosphorylated AKT (p-AKT, D ) and AKT ( E ) protein abundance in ipsilateral L4/5 DRG tissues obtained from vehicle and IGF1-treated naïve rats. n = 6 rats per group. C , representative blots are shown. F and G , western blotting analysis of HDAC2 protein abundance in ipsilateral L4/5 DRG tissues obtained from vehicle and IGF1-treated naïve rats. n = 6 rats per group. H – J , RT-qPCR analysis and western blotting analysis of ClC-3 mRNA and protein abundance in L4/5 DRG tissues from vehicle and IGF1-treated naïve rats. n = 4 to 8 rats per group. K and L , assessment of the PWT ( K ) and PWL ( L ) for naïve rats that received intrathecal IGF1 or vehicle, performed at 5 days after drug administration (n = 10 rats per group). M , assessment of animal’s locomotor function before and after intrathecal drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( J ); two-way ANOVA with Sidak’s post hoc test for ( K )–( M ). BCP, bone cancer pain; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; IGF1, insulin-like growth factor 1; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Igf1r in DRG neurons disrupts intrathecal IGF1-induced activation of AKT signaling as well as the upregulated expression of HDAC2, the decreased expression of ClC-3 protein and pain hypersensitivity in na9;ve rats . A and B , RT-qPCR analysis and western blotting analysis of IGF1R mRNA and protein abundance in L4/5 DRG tissues from sham and BCP rats at 14 days after tumor cells inoculation. n = 6–7 rats per group. C , representative images showing the colocalization of IGF1R, HDAC2, and ClC-3 in DRG neurons. The scale bar represents 25 μm. D – F , western blotting analysis of p-AKT ( E ) and AKT ( F ) protein abundance in ipsilateral L4/5 DRG tissues obtained from IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP, respectively (n = 4 rats per group). D , representative blots are shown. G – I , western blotting analysis of HDAC2 ( H ) and ClC-3 ( I ) protein abundance in ipsilateral L4/5 DRG tissues obtained from IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP, respectively (n = 4 rats per group). G , representative blots are shown. J and K , assessment of ipsilateral PWT and PWL for IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP (n = 10 rats per group). L , assessment of animal’s locomotor function before and after intrathecal drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( B ); one-way ANOVA followed by Dunnett's post hoc test for ( E )–( I ) two-way ANOVA with Sidak’s post hoc test for ( J )–( L ). BCP, bone cancer pain; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; IGF1, insulin-like growth factor 1; LV-GFP, lentivirus expressing green fluorescent protein; p-AKT, phosphorylated AKT; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Igf1r in DRG neurons impairs HDAC2-mediated transcriptional repression of Clc-3 gene, reduces neuronal excitability and attenuates pain hypersensitivity in bone metastasis model rats . A – E , western blotting analysis of p-AKT ( B ), AKT ( C ), HDAC2 ( D ) and ClC-3 ( E ) abundance in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shIGF1R treated rats at 14 days after tumor cells inoculation. n = 4 to 6 rats per group. A , representative blots are shown. F – H , electrophysiological analyses of neuronal excitability in ipsilateral L4/5 DRG neurons of bone metastasis model rats that received intrathecal LV-GFP and LV-shIGF1R, recorded at 14 days after tumor cells inoculation. F , representative neuronal action potentials evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. The scale bar represents 20 mV, 100 ms. G and H , plots of threshold potential and rheobase are shown. n = 10∼20 cells from three rats per group. I and J , assessment of ipsilateral PWT ( I ) and PWL ( J ) from intrathecal LV-GFP and LV-shIGF1R treated rats after tumor cells inoculation. n = 10 rats per group. K , assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration. n = 10 rats per group. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( B )–( H ); two-way ANOVA with Sidak’s post hoc test for ( I )–( K ). DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; LV-GFP, lentivirus expressing green fluorescent protein; p-AKT, phosphorylated AKT; PWL, paw withdrawal latency; PWT, paw withdrawal threshold.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Histone Deacetylase Assay, Expressing