Journal: Neuron

Article Title: The sodium-bicarbonate cotransporter Slc4a5 mediates feedback at the first synapse of vision

doi: 10.1016/j.neuron.2024.08.015

Figure Lengend Snippet: Slc4a5 knockout mice lack horizontal cell feedback (A) Left: GCaMP6s was targeted to cones by subretinal injection of BP2 serotype AAV-ProA1-GCaMP6s. Right: schematic of the retina. Cones, green; horizontal cells, red. Dashed line, focal plane of two-photon imaging. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Two visual stimulation approaches to evoke horizontal cell feedback. Schematics of the time courses, the spatial configurations of the stimuli, and the expected responses of the cones in the imaging area are shown. Left: stimulations with small or large spots. Right: stimulation with an annulus. (C) Two-photon images of cone axon terminals expressing GCaMP6s in wild-type (WT), Slc4a3 tm1 , and Slc4a5 tm1 mice. (D) Small (gray) and large (black) spot stimulation response traces of GCaMP6s fluorescence in a single cone terminal in no-drug control (left) and in NBQX (right). (E) Left: scatterplot of cone responses to a large spot (R C+S ) and a small spot (R C ) in no-drug control (blue) and in NBQX (red). Right: histogram of the difference between large and small spot responses (R C+S − R C ) in no-drug control (blue) and in NBQX (red). (F) Annulus stimulation response traces of GCaMP6s fluorescence in a single cone terminal in no-drug control (left) and in NBQX (right). The traces are from the same cells as shown in (D). (G) Left: scatterplot of cone responses to an annulus (R S ) and to a small spot (R C ) in no-drug control (blue) and in NBQX (red). Right: histogram of annulus response (R S ) in no-drug control (blue) and in NBQX (red). (H) Left: EGFP was targeted to horizontal cells by intravenous injection of PHP.eB serotype AAV-ProA445-EGFP. Right: schematic of two-photon guided sharp electrode recording of horizontal cells. (I) Confocal images of a retinal section from a mouse injected with PHP.eB AAV-ProA445-EGFP. Calbindin was immunostained as a marker for horizontal cells (magenta). Höchst was used to stain the nuclei (gray). Arrowheads, horizontal cell somas. (J) Two-photon images of the EGFP labeled horizontal cells (green) and the recorded dye-filled horizontal cell (red). Arrows indicate the sharp electrode filled with Alexa 568 dye. (K) Full-field stimulation response traces of membrane voltages in a horizontal cell in wild-type (WT) and Slc4a5 tm1 mice. (L) Response peak amplitude and resting potential of horizontal cells in wild-type (WT) and Slc4a5 tm1 mice. Two outliers are removed from the peak amplitude plot. (M) The proposed Slc4a5-dependent mechanism of light-evoked horizontal cell feedback. VGCC, voltage-gated calcium channel; Glu, glutamate; In, intracellular space; Ex, extracellular space. Illustrations were partly created by BioRender. See also Figures S1 – .

Article Snippet: Sections were then incubated overnight at 4 °C with a mouse monoclonal anti-Calbindin primary antibody (1:1000; Swant, #300PUR) diluted in 200 μL of blocking buffer B (see above).

Techniques: Knock-Out, Injection, Imaging, Expressing, Fluorescence, Control, Marker, Staining, Labeling, Membrane

Journal: Neuron

Article Title: The sodium-bicarbonate cotransporter Slc4a5 mediates feedback at the first synapse of vision

doi: 10.1016/j.neuron.2024.08.015

Figure Lengend Snippet: Horizontal-cell-specific knockdown of Slc4a5 in adult mice (A) Horizontal-cell-specific targeting of shRNAs against Slc4a5 and simultaneous labeling of cones with GCaMP6s. Schematic of the retina shows horizontal cells labeled with tdTomato (red) and cones with GCaMP6s (green). Dashed line, focal plane of two-photon imaging. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Confocal images of retinal sections from mice injected as in (A), stained for GCaMP6s, Calbindin, and tdTomato with anti-GFP, anti-Calbindin, and anti-RFP antibodies, respectively. Cell nuclei were labeled with Höchst. (C) Two-photon images of cone axon terminals expressing GCaMP6s in Cre −/− control and Cre +/− Slc4a5 knockdown mice. (D) Small (gray) and large (black) spot stimulation response traces of GCaMP6s fluorescence in a single cone terminal in no-drug control (left) and in NBQX (right). (E) Left: scatterplot of cone responses to a large spot (R C+S ) and a small spot (R C ) in no-drug control (blue) and in NBQX (red). Right: histogram of the difference between large and small spot responses (R C+S − R C ) in no-drug control (blue) and in NBQX (red). (F) Annulus stimulation response traces of GCaMP6s fluorescence in a single cone terminal in no-drug control (left) and in NBQX (right). The traces are from the same cells as shown in (D). (G) Left: scatterplot of cone responses to an annulus (R S ) and to a small spot (R C ) in no-drug control (blue) and in NBQX (red). Right: histogram of annulus response (R S ) in no-drug control (blue) and in NBQX (red). See also and .

Article Snippet: Sections were then incubated overnight at 4 °C with a mouse monoclonal anti-Calbindin primary antibody (1:1000; Swant, #300PUR) diluted in 200 μL of blocking buffer B (see above).

Techniques: Knockdown, Labeling, Imaging, Injection, Staining, Expressing, Control, Fluorescence

Journal: Neuron

Article Title: The sodium-bicarbonate cotransporter Slc4a5 mediates feedback at the first synapse of vision

doi: 10.1016/j.neuron.2024.08.015

Figure Lengend Snippet:

Article Snippet: Sections were then incubated overnight at 4 °C with a mouse monoclonal anti-Calbindin primary antibody (1:1000; Swant, #300PUR) diluted in 200 μL of blocking buffer B (see above).

Techniques: Virus, Recombinant, RNAscope, Multiplex Assay, Expressing, Sequencing, Positive Control, Negative Control, Software, Imaging

Journal: Cancers

Article Title: Brief Magnetic Field Exposure Stimulates Doxorubicin Uptake into Breast Cancer Cells in Association with TRPC1 Expression: A Precision Oncology Methodology to Enhance Chemotherapeutic Outcome

doi: 10.3390/cancers16223860

Figure Lengend Snippet: Immunohistochemistry (IHC) staining of breast tumor and neighboring normal breast tissue. ( A ) Representative IHC images showing TRPC1 and Ki-67 staining (brown) in breast tumor (Grade 1 and 3) and neighboring normal breast tissue. Tissue sections were counterstained with hematoxylin (blue). The scale bar is 50 μm. ( B ) Semi-quantitative IHC analysis of TRPC1 and Ki-67 staining intensity. Staining intensity was scored as: 0 (no staining), 1 (weak), 2 (moderate) or 3 (strong staining). Data represent mean ± standard error of the mean (SEM). The number of independent samples is reflected within each bar, and the samples were analyzed using one-way ANOVA, followed by Šidák’s multiple comparison post hoc test. Statistical significance is indicated by **, p ≤ 0.01 and ***, p ≤ 0.001.

Article Snippet: Primary monoclonal mouse TRPC1 antibody (E-6) (1:50; catalogue no.: sc-133076; Santa Cruz Biotechnology, Dallas, TX, USA) and monoclonal rabbit antigen Kiel 67 (Ki-67) antibody (1:400; catalogue no.: #9129; Cell Signaling Technologies, Danvers, MA, USA) were used.

Techniques: Immunohistochemistry, Staining, Comparison

Journal: Cancers

Article Title: Brief Magnetic Field Exposure Stimulates Doxorubicin Uptake into Breast Cancer Cells in Association with TRPC1 Expression: A Precision Oncology Methodology to Enhance Chemotherapeutic Outcome

doi: 10.3390/cancers16223860

Figure Lengend Snippet: Magnetic field-induced DOX uptake correlates with TRPC1 expression. ( A ) Bar chart showing the fold change in intracellular DOX concentration of 4T1 cells pre-treated with 50 µM SKF-96365 for 15 min and 500 nM DOX for 5 min prior to 10 min magnetic exposure (n = 4). ( B ) Bar chart showing fold changes in TRPC1 transcript levels as detected by qPCR in 4T1 cells transfected with scrambled or TRPC1 dsiRNA after 24 h. Data represent mean ± standard deviation, (n = 3 technical replicates). ( C ) Bar chart showing fold change in intracellular DOX concentration in 4T1 cells transfected with scramble or TRPC1 dsiRNA. Cells were pre-treated with 500 nM DOX for 5 min prior to 10 min magnetic exposure (n = 3). ( D ) Bar chart showing fold change in TRPC1 transcript level detected by qPCR in MCF7 and MCF7 stable cell line overexpressing TRPC1 (MCF7-TRPC1) cells (n = 3). ( E ) Western blot of GFP-TRPC1 in MCF7 and MCF7-TRPC1 cells (n = 4). The uncropped blots are shown in . ( F ) Bar chart showing the fold change in intracellular DOX concentration of MCF7 and MCF7-TRPC1 cells pre-treated with 500 nM DOX for 5 min prior to 10 min of magnetic exposure (n = 4). Unless otherwise stated, data represent mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA, followed by Šidák’s multiple comparison post hoc test. Significance is indicated by ns (not significant); **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001.

Article Snippet: Primary monoclonal mouse TRPC1 antibody (E-6) (1:50; catalogue no.: sc-133076; Santa Cruz Biotechnology, Dallas, TX, USA) and monoclonal rabbit antigen Kiel 67 (Ki-67) antibody (1:400; catalogue no.: #9129; Cell Signaling Technologies, Danvers, MA, USA) were used.

Techniques: Expressing, Concentration Assay, Transfection, Standard Deviation, Stable Transfection, Western Blot, Comparison