Journal: The Journal of Biological Chemistry

Article Title: Tamoxifen metabolites acting via BK Ca orchestrate the dynamics of K + and Ca 2+ in breast cancer cells

doi: 10.1016/j.jbc.2025.111015

Figure Lengend Snippet: TAM + M trigger cytosolic K + dynamics in BC cells independently of ER . ( A , B , C , F , and G ), K + FRET-ratio signals obtained by live-cell imaging of cytosolic K + concentrations ([K + ] i ) over time of hBK Ca low (ER + or ER - ), hBK Ca medium (ER + ) and hBK Ca high (ER - ) BC cells and ( A–G ) corresponding bar charts with individual data points for statistical analysis, which is based on the difference of the basal (R 0 ) to the minimal (R) K + FRET/CFP ratio signals and expressed as Δ Min (R/R 0 ). Alterations of [K + ] i were induced by bath-administration of ( A , B ) BK Ca activator NS11021 (hBK Ca low from A adapted in B ), active TAM metabolites ( C ) 4-OH-TAM and ( D , E , G ) endoxifen or ( F ) E2 following 10 min of FRET recording at basal conditions. ER + hBK Ca low cells expressed in addition either ( A , G ) RFP (+RFP), RFP-tagged BK Ca (+BK Ca -RFP rescue ) or RFP-tagged “pore-death” BK Ca G354S (+BK Ca G354S ) as indicated. FRET-ratio signal responses are displayed for ( C , E , G ) 1 μM 4-OH-TAM or endoxifen and ( F ) 1 μM E2. Data represents average ± SD. n(independent experiments) = ( A ) 4-5, ( B ) 5-7, ( C ) 3-6, ( D ) 4-6, ( E ) 4-6, ( F ) 3-4, ( G ) 5 with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. ( A , B , E , G ) One-way ANOVA followed by Tukey‘s multiple comparisons test and ( C , D , F ) Two-way ANOVA followed by Tukey‘s multiple comparisons test with significant group differences in ( C , D ). Statistical values are described in detail in .

Article Snippet: Loss-of-function mutant BK Ca G354S previously reported by , was generated by site-directed mutagenesis using: BK Ca -RFP plasmid as template, the primers: BK G354S forward 5′-ACA ATG TCT ACA GTG AGT TAT GG-3′ and BK G354S reverse 5′-CAT AAA CGT CCC CAT AAC TCA-3′, Q5 High-Fidelity DNA Polymerase (NEB) followed by digestion with Dpn I (NEB) according to manufacturer’s instructions.

Techniques: Live Cell Imaging

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Il1a , Ccl5 , and Ccl1 mRNA levels in the dorsal horn of female mice. n = 5 to 8. ( B ) Immunofluorescence image of IL-1α (green) in vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). ( C ) Double immunofluorescence staining of CCL1 (green) and vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). ( D and E ) Coimmunostaining image of F4/80 (green) and CD31 (red) in the spinal dorsal horn and the statistical graph. Scale bar, 200 μm. n = 5. ( F ) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. ( G ) Immunofluorescence image shows IL-1R1 (red) expression in macrophages (green). Scale bar, 10 μm. ( H and I ) Intrathecal IL-1R1 injection inhibited the up-regulation of F4/80 (green) and CD31 (red) in the near vascular of the spinal dorsal horn. Scale bar, 200 μm (H). The number of F4/80 high cells in the dorsal horn of the spinal cord [(I), left]. The percentage of F4/80 high area among CD31 + area [(I), right]. n = 4 to 5. ( J and K ) Immunostaining shows F4/80 (green) and CD31 (red) expression in the spinal dorsal horn. Scale bar, 200 μm (J). The number of F4/80 high cells in the dorsal horn of the spinal cord [(K), left]. The percentage of F4/80 high area among CD31 + area [(K), right]. n = 5. ( L ) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. Significance: * P < 0.05, # P < 0.05, ** P < 0.01, and ## P < 0.01.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Immunofluorescence, Double Immunofluorescence Staining, Injection, Expressing, Immunostaining

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Coimmunofluorescence of CCL1 (green) and macrophages (purple) on day 3 following BTZ treatment in female mice. Scale bar, 50 μm. ( B ) Quantification of CCL1 fluorescence intensity in macrophages. n = 5. ( C ) Coimmunofluorescence of CCR8 (red) and NeuN (marker of neuron, green). Scale bars, 20 μm. ( D ) Coimmunofluorescence of CCR8 (red) and glial fibrillary acidic protein (GFAP; a marker of astrocyte, green). Scale bars, 20 μm. ( E ) Representative traces (left) and statistical data (right) for the action potential firing recorded in spinal cord lamina II neurons. n = 4 mice. ( F ) Intrathecal injection of CCL1-neutralizing antibody attenuated the mechanical allodynia induced by BTZ. n = 5 to 7. ( G ) CCL1 increased the action potentials of neurons in female mouse spinal slices. n = 6. ( H ) Intraspinal CCL1 injection reduced the mechanical withdrawal threshold. n = 5. ( I ) R243 preincubation blocked the CCL1-induced action potential increase in female mouse spinal slices. n = 6. ( J ) Intrathecal injection of R243 attenuated the mechanical allodynia induced by BTZ. n = 5. ( K ) Intrathecal injection of R243 attenuated the mechanical allodynia induced by recombinant CCL1. n = 5. ( L ) Recombinant CCL1 increased the action potentials in astrocyte CCR8 knockdown mouse spinal slices. n = 5. ( M ) Intraspinal CCL1 injection reduced the mechanical withdrawal threshold in astrocyte CCR8 knockdown female mice. n = 5. Significance: not significant (n.s.), * P < 0.05 and ** P < 0.01.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Fluorescence, Marker, Injection, Recombinant, Knockdown

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Immunofluorescence imaging showed BTZ-activated astrocytes (turquoise) in close proximity to macrophages (green) and vascular endothelial cells (red). Scale bars, 20 μm. ( B ) Schematic of two-photon imaging configuration. ( C ) Representative image showed the increased GCaMP6s (green) in spinal cord astrocytes (SR101, red) following CCL1 incubation. Scale bar, 150 μm. ( D ) Example signal trace (left) and histogram of area under the curve (AUC) (right) of calcium signaling value from the spinal dorsal horn astrocyte in mice treated with CCL1. n = 3. ( E to G ) R243 incubation prevented CCL1-induced calcium signaling increase in GCaMP6s + astrocytes. Representative image (E) and signal trace (F) show R243 blocked CCL1-induced calcium up-regulation. Scale bar, 20 μm. AUC quantification (G). n = 3. ( H to J ) Response of astrocytes to recombinant CCL1 during bath application of TTX (1 μM). Representative image (H) and signal trace (I) showed that TTX did not inhibit the increase in intracellular calcium signaling induced by CCL1. Scale bar, 20 μm. AUC quantification (J). n = 3. ( K to M ) Application of CNO increased the astrocyte intracellular calcium signaling in the female with injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. Scale bar, 20 μm. ( N ) The action potential number in female mouse spinal cord slice was increased by CNO application. Representative trace (left) and statistical data (right) were shown. n = 3. ( O ) Application with CNO decreased the paw withdrawal threshold in female mice with intraspinal injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. n = 5. Significance: not significant (n.s.) and * P < 0.05.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Immunofluorescence, Imaging, Incubation, Recombinant, Injection

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Increased ICAM-1 promotes monocyte adhesion to vascular endothelial cells following BTZ treatment. ( B ) IL-1α/ICAM-1 collaboratively promote monocyte transmigration across the vascular wall. ( C ) Infiltrated macrophage-released CCL1 enhances neuron excitability directly or indirectly via the activated astrocytes.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Transmigration Assay