quercetin, antioxidative flavonoid  (Advisains)

Journal: Nature Communications

Article Title: USP36 stabilizes nucleolar Snail1 to promote ribosome biogenesis and cancer cell survival upon ribotoxic stress

doi: 10.1038/s41467-023-42257-8

Figure Lengend Snippet: a Immunofluorescence staining assays were performed to examine Snail1 or nucleolus marker B23 in either fixed HCC1806 cells, frozen sections of A549 cell-derived xenograft tumor (A549-CDX), or paraffin sections of two clinical breast tumor samples. Notice that Snail1 was accumulated in the nucleolus in a few cells. b , c HCC1806 cells were treated with ribosome inhibitor HHT (20 ng/mL and hereafter), a Pol I inhibitor CX-5461 (200 nM), mTOR inhibitor rapamycin (Rapa, 20 nM), or ER stress inducer Tunicamycin (Tunica, 2 μg/mL) for 24 h. Cells were subjected to immunofluorescence staining analyses ( b ). The co-localization between Snail1 and B23 (as analyzed by Pearson’s correlation coefficient , ) was quantified and statistically analyzed ( c ). d , e Hs 578T, SUM159, or A549 cells were treated with or without HHT for 24 h. Cells were subjected to immunofluorescence staining analyses ( d ). The co-localization between Snail1 and B23 was quantified and statistically analyzed ( e ). f , g HCC1806 cells were treated with a ribotoxic inducer anisomycin (Aniso, 50 ng/mL), puromycin (Puro, 200 ng/mL), G418 (1 μg/mL), or blasticidin (Blasti, 2 μg/mL) for 24 h. Cells were subjected to immunofluorescence staining analyses ( f ). The co-localization between Snail1 and B23 was quantified and statistically analyzed ( g ). h HCC1806 cells were treated with or without HHT for 24 h followed by cell fractionation and western blot analyses. CF Cellular fraction, CP Cytoplasm, NP Nucleoplasm, No Nucleolus. This experiment has been repeated for three times with similar results. Quantification of the co-localization between Snail1 and B23 using Pearson’s correlation coefficient ( c , e , g ). 40 cells derived from three independent experiments were randomly chosen and subjected to quantification analyses. Data were presented as mean ± SD. Comparisons were performed with unpaired two-tailed Student’s t test. Scale bar, 25 μm.

Article Snippet: Tunicamycin (ab120296) was purchased from Abcam (Cambridge, MA, USA).

Techniques: Immunofluorescence, Staining, Marker, Derivative Assay, Cell Fractionation, Western Blot, Two Tailed Test

Journal: Lipids in Health and Disease

Article Title: Sirt6 enhances macrophage lipophagy and improves lipid metabolism disorder by regulating the Wnt1/β-catenin pathway in atherosclerosis

doi: 10.1186/s12944-023-01891-3

Figure Lengend Snippet: Autophagy attenuates the area of necrotic core in plaques and inhibits the formation of foam cells. ApoE −/− , ApoE −/− + Rap and ApoE −/− : ATG5 −/− mice were fed for 16 weeks with western diet ( n = 10) to trigger atherosclerosis. A Lipids in the aortic root were stained with oil red O. B Assessment of the necrotic area quantitatively. Data are expressed as mean ± SE (One way ANOVA showed that there was statistical significance among groups. * P < 0.05 vs. ApoE −/− mice [ n = 10]; # P < 0.05 vs. ApoE −/− + Rap on a western diet [ n = 10]; Student’s t-test showed that there was statistical significance between ApoE −/− + Rap and ApoE −/− mice, P < 0.01; in addition, there was statistical significance between ApoE −/− : ATG5 −/− mice and ApoE −/− mice, P < 0.01). C – E Autophagy inhibited the conversion of macrophages into foam cells. The cells were activated by 50-μg/mL Ac-LDL in the presence of rapamycin, 3-MA, siRNA Atg5 and siRNA Atg7 for 30 h and subsequently stained with oil red O. D , F Quantitative analysis of the intensity of oil red O staining. Data are presented as mean ± SEM (One way ANOVA showed that there was statistical significance among groups. * P < 0.05 vs. the Ac-LDL group; # P < 0.05 vs. the Ac-LDL + Rap and Ac-LDL + siRNA Atg5 groups [ n = 6 independent experiments])

Article Snippet: After treating macrophages with Ac-LDL for 30 h, they were subsequently treated with rapamycin (ab120224, Abcam) to promote autophagy or exposed to 3-MA (Sigma-Aldrich Shanghai Trading Co., Ltd.) to inhibit autophagy.

Techniques: Western Blot, Staining

Journal: Nature Communications

Article Title: Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary–lung axis

doi: 10.1038/s41467-023-41319-1

Figure Lengend Snippet: a Schematic diagram of the experimental design (6–8 weeks, female mice). b α-MSH in the serum were measured by ELISA ( n = 6/group). c qRT‒PCR analysis of Crh expression in the hypothalamus ( n = 3/group). d Representative CRF staining in the hypothalamus ( n = 3/group) (CRH: red; DAPI: blue; scale bar, 50 μm). Representative CRH quantification in the hypothalamus. e Proteins were comigrated by SDS–PAGE with homogenized hypothalamic tissue and visualized by Western blot analysis with anti-mouse CRH antibodies. Statistical analysis of CRH expression was performed using ImageJ ( n = 3/group). f qRT‒PCR analysis of Pomc expression in the hypothalamus, skin, and pituitary gland ( n = 3, 2, 3, 3, 3, 3 from left to right). g Melanocortin peptides (shaded boxes) derived from POMC. h qRT‒PCR analysis of Pcsk1 and Pcsk2 expression in the pituitary gland ( n = 3/group). i Representative POMC staining in the pituitary gland (POMC: green; DAPI: blue; scale bar, 50 μm). j Proteins were comigrated by SDS–PAGE with homogenized pituitary gland tissue and visualized by Western blot analysis with anti-mouse POMC antibodies. Statistical analysis of POMC expression was performed using ImageJ ( n = 3/group). k Flow cytometric analysis of IL-5 and IL-13 in lung ILC2s 3 days after α-MSH/β-endorphin treatment stimulated with PMA plus ionomycin and BFA for 4 h ( n = 3, 6, 6, 6 from left to right). l ELISA was performed to measure IL-5 and IL-13 in ILC2s culture medium after 3 days of treatment with α-MSH/β-endorphin was detected by ELISA ( n = 2, 6, 6, 6 from left to right). Lung ILC2s sorted from IL-33-treated WT female mice were cultured in vitro with IL-2 (100 U/ml), mIL-7 (20 ng/ml), and mIL-33 (1 ng/ml) in the presence or absence of α-MSH/β-endorphin. Each symbol represents an individual mouse ( b–f , h , j ). Gene expression was analyzed by qRT-PCR and normalized with Hprt using the 2 -△△Ct method ( c , f , h ). The data are representative of at least three independent experiments. The bars and error bars show the means ± SDs. For statistical analysis, the following tests were used. b , k , l one-way ANOVA with Tukey’s multiple comparisons test. c–f , h , j Two-tailed unpaired Student’s t -test.

Article Snippet: On day 26, the mice were sacrificed for analysis. α-MSH (Abcam, ab120205, 4 mg/kg) or an α-MSH release inhibitor (Tocris, 1929, 1 mg/kg) was intratracheally (i.t.) or intraperitoneally (i.p.) administered according to the experimental design.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Staining, SDS Page, Western Blot, Derivative Assay, Cell Culture, In Vitro, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary–lung axis

doi: 10.1038/s41467-023-41319-1

Figure Lengend Snippet: a R5 /+ and Mc5r fl/fl R5 /+ female mice (6–8 weeks) were intratracheally challenged with papain (4 µg/mouse) or PBS for 5 consecutive days with or without UVB irradiation of the eyes 24 h before day 1 and then sacrificed on day 6. b Percentage of eosinophils in the BALF ( n = 6/group). c Representative H&E staining of lung sections (bars, 200 μm) ( n = 6/group). d IL-5 and IL-13 in the BALF were detected by ELISA ( n = 6/group). e Statistical analysis of the numbers of ILC2s in the lungs ( n = 6/group). f Flow cytometric analysis of the percentages of IL-5 + and IL-13 + cells among lung ILC2s stimulated with PMA plus ionomycin and BFA for 4 h ( n = 6/group). g WT female mice (6–8 weeks) were intratracheally challenged with papain (4 µg/mouse) or PBS for 5 consecutive days, treated with PBS or α-MSH (5 mg/kg) daily, and sacrificed on day 6. h Quantification of the eosinophil percentage in the BALF ( n = 7, 6, 6, 6 from left to right). i Representative H&E staining of lung sections (bars, 200 μm) ( n = 6/group). j ELISA was performed to measure the levels of IL-5 and IL-13 in the BALF ( n = 7, 7, 6, 6 from left to right). k Statistical analysis of the numbers of ILC2s in the lungs ( n = 7, 6, 6, 6 from left to right). l Flow cytometric analysis of IL-5 and IL-13 in lung ILC2s stimulated with PMA plus ionomycin and BFA for 4 h ( n = 7, 6, 6, 6 from left to right). m ILC2s from R5 /+ and Mc5r fl/fl R5 /+ female mice cultured with the culture supernatant of pituitary cells from WT female mice(6-8w) treated with or without UVB irradiation to the eyes. n α-MSH in the pituitary cell culture supernatant was measured by ELISA ( n = 4/group); IL-5 and IL-13 in the supernatants were detected by ELISA ( n = 4/sample). Each symbol represents an individual mouse ( b–h , j – m ). The data are representative of at least three independent experiments. The bars and error bars show the means ± SDs. For statistical analysis, the following tests were used. b–h , j – m Two-tailed unpaired Student’s t -test.

Article Snippet: On day 26, the mice were sacrificed for analysis. α-MSH (Abcam, ab120205, 4 mg/kg) or an α-MSH release inhibitor (Tocris, 1929, 1 mg/kg) was intratracheally (i.t.) or intraperitoneally (i.p.) administered according to the experimental design.

Techniques: Irradiation, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary–lung axis

doi: 10.1038/s41467-023-41319-1

Figure Lengend Snippet: a–e Lung ILC2s were sorted from R5 /+ and Mc5r fl/fl R5 /+ female mice (6–8 weeks), and transcriptional profiling was performed. a GSEA of the asthma pathway. NES, normalized enrichment score. b Heatmap of asthma-characteristic genes in ILC2s. c Heatmap of known ILC2 regulators. d Volcano plot of differentially expressed genes (log2(fold change) > 2; P < 0.05) in ILC2s compared between R5 /+ and Mc5r fl/fl R5 /+ mice. The ILC2 regulators mentioned in D are highlighted. e Heatmap of selected genes in JAK/STAT signaling pathways. f MFIs of CD127 in ILC2s ( n = 3, 2, 3, 2 from left to right). g ELISA was performed to measure IL-5 and IL-13 secretion into the mouse ILC2 culture medium after 3 days ( n = 3/group). Lung ILC2s sorted from IL-33-treated WT female mice were cultured with IL-2 (100 U/ml), mIL-33 (1 ng/ml), and IL-7 (0, 2, 20, 200 pg/ml, 2, 20, and 200 ng/ml) ± α-MSH. h Sorted lLC2s were cultured ± α-MSH for 3 days, then cultured in the absence of cytokines for 24 h and re-stimulated with IL-7 for 0, 10, and 60 min for Western blot analysis (up); the levels p-STAT3 and p-STAT5 were performed using ImageJ (down) ( n = 2/group). i IL-5 and IL-13 in ILC2 culture medium were detected by ELISA after 3 days ( n = 4/group). ILC2s cultured with IL-2 (100 U/ml), IL-7(20 ng/ml), or IL-33(1 ng/ml), respectively, ±α-MSH. j IL-5 and IL-13 were detected by ELISA after 3 days. ILC2s cultured in combinations of IL-2 (100 U/ml) plus IL-33(1 ng/ml), IL-7(20 ng/ml) plus IL-33(1 ng/ml), or IL-2 (100 U/ml) plus IL-7(20 ng/ml) and IL-33 (1 ng/ml) ±α-MSH ( n = 4/group). k Sorted lLC2s were cultured ±α-MSH for 3 days, then cultured in the absence of cytokines for 24 h and re-stimulated with IL-7, IL-33, IL-7 plus IL-33 for 0, 10, and 60 min for Western blot analysis ( n = 2/group). The data are representative of two or more independent experiments. The bars and error bars show the means ± SDs. g , i , j Two-tailed unpaired Student’s t -test.

Article Snippet: On day 26, the mice were sacrificed for analysis. α-MSH (Abcam, ab120205, 4 mg/kg) or an α-MSH release inhibitor (Tocris, 1929, 1 mg/kg) was intratracheally (i.t.) or intraperitoneally (i.p.) administered according to the experimental design.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary–lung axis

doi: 10.1038/s41467-023-41319-1

Figure Lengend Snippet: a WT female mice (6–8 weeks) were challenged for 3 consecutive days with HDM (30 µg/mouse) and treated with PBS or α-MSH (4 mg/kg) daily. Then, the mice were allowed to rest for 10 days, rechallenged with HDM (6 µg/mouse) for 4 consecutive days, and sacrificed on day 18. b The total IgE levels, HDM-specific IgE, and HDM-specific IgG1 levels in the serum of PBS- and α-MSH-treated mice were measured by ELISA ( n = 7, 8); the antibody titers were extrapolated from a standard curve (if available), or the results are presented as the absorbance values at 450 nm (OD450 values) for the serum dilution for which all samples were in the range of the assay. c Line graphs show lung resistance in response to increasing doses of methacholine ( n = 2 mice for PBS,4 mice for HDM, 4 mice for HDM+α-MSH). d Percentage and number of eosinophils in the BALF ( n = 7/group). e The levels of IL-5 and IL-13 in the BALF were measured by ELISA ( n = 7, 8). f Representative H&E and PAS staining of lung sections (bars, 200 μm) ( n = 7, 8). g The number of ILC2s in the lungs ( n = 7, 8). h The number of IL-5 + IL-13 + cells among lung ILC2s after stimulation with PMA, ionomycin, and BFA for 4 h ( n = 7, 8). i The number of ST2 + CD4 + T cells in the lungs ( n = 7, 8). j The number of IL-5 + IL-13 + cells among lung ST2 + CD4 + T cells after stimulation with PMA, ionomycin, and BFA for 4 h ( n = 7, 8). Each symbol represents an individual mouse ( b–h , j ). The bars and error bars show the means ± SDs. b–e , g–j Two-tailed unpaired Student’s t -test.

Article Snippet: On day 26, the mice were sacrificed for analysis. α-MSH (Abcam, ab120205, 4 mg/kg) or an α-MSH release inhibitor (Tocris, 1929, 1 mg/kg) was intratracheally (i.t.) or intraperitoneally (i.p.) administered according to the experimental design.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary–lung axis

doi: 10.1038/s41467-023-41319-1

Figure Lengend Snippet: a MC5R expression in hILC2s (FVD − CD45 + LIN − CD161 + CD127 + CRTH2 + ) and CD4 + T cells and B cells sorted from human PBMCs ( n = 2/group). MC-R expression in hILC2s sorted from PBMCs ( n = 2/group). Gene expression was analyzed by qRT-PCR and normalized with hGAPDH using the 2 -△△Ct method. b hIL-5 and hIL-13 in hILC2s culture medium were detected by ELISA after 5 days of treatment with α-MSH ( n = 3/group). hILC2s were cultured with hIL-2 (100 U/ml), hIL-7 (20 ng/ml), and hIL-33 (25 ng/ml) ±α-MSH. c Correlation analysis between the percentages and numbers of hILC2s among PBMCs and α-MSH concentration in the plasma of APs ( n = 34). d Correlation analysis between IL-5/IL-13 and the α-MSH concentration in the plasma of APs ( n = 34). e Secretion of IL-5 and IL-13 by PBMCs after IL-33 activation ± α-MSH ( n = 34). PBMCs from APs cultured with hIL-2 (100 U/ml), hIL-7 (20 ng/ml), and hIL-33 (50 ng/ml). IL-5 and IL-13 in supernatants were measured by ELISA on day 5. PBMCs with one-fold higher IL-5 secretion after IL-2/IL-7/IL-33 treatment than after IL-2/IL-7 treatment were used for data analysis. f The WT female mice (6–8 weeks) were intratracheally challenged with papain for 5 consecutive days and intratracheally treated with PBS or α-MSH (4 mg/kg) for two consecutive days and sacrificed on day 10. g Flow diagram showing the percentage of eosinophils in the BALF. h Percentage and number of eosinophils in the BALF ( n = 8, 7 from left to right). i HE and PAS staining of lung sections (bars, 200 μm) ( n = 8, 7 from left to right). j Statistical analysis of the numbers of lung ILC2s ( n = 7/group). k Flow cytometric analysis of IL-5 and IL-13 in lung ILC2s stimulated with PMA plus ionomycin and BFA for 4 h. l Statistical analysis of the percentages of IL-5 + and IL-13 + cells among lung ILC2s ( n = 7/group). Each symbol represents an individual patient ( c–e ) or mouse ( h , j–l ). The bars and error bars show the means ± SDs. For statistical analysis, the following tests were used. c , d Pearson’s correlation coefficient analysis. b , h , j , l Two-tailed unpaired Student’s t -test. e one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: On day 26, the mice were sacrificed for analysis. α-MSH (Abcam, ab120205, 4 mg/kg) or an α-MSH release inhibitor (Tocris, 1929, 1 mg/kg) was intratracheally (i.t.) or intraperitoneally (i.p.) administered according to the experimental design.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Activation Assay, Staining, Two Tailed Test

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: NEK7 inhibition attenuates Aβ 42 -induced cognitive impairment by regulating TLR4/NF-κB and the NLRP3 inflammasome in mice

doi: 10.3164/jcbn.22-105

Figure Lengend Snippet: Silencing of NEK7 attenuates the inflammation of Aβ 42 -induced BV2 cells. BV2 cells were transfected with si-RNA or si-NEK7 for 24 h, and then treated with Aβ 42 (200 ng/ml) for 12 h. (A–C) The levels of IL-6, IL-8, and TNF-α in BV2 cells were measured by ELISA assay. (D) The protein expression of iNOS and COX-2 was detected by Western blot. (E) The protein expression of BDNF and NGF was detected by Western blot. (F) Western blot was used to measure the protein expression of iNOS, COX-2, BDNF, and NGF. * means compared with untreated group p <0.05, ** means compared with untreated group p <0.01, and # means compared with si-RNA group p <0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Article Snippet: Blots were probed with the following antibodies: rabbit polyclonal to GAPDH-Loading Control (1:1,000, abcam, ab8245), rabbit polyclonal anti-NEK7 antibody (1:2,000, abcam, ab133514), rabbit monoclonal anti-caspase-3 antibody (1:2,000, abcam, ab32351), rabbit monoclonal anti-caspase-9 antibody (1:10,000, abcam, ab65608), rabbit monoclonal anti-Bax antibody (1:1,000, abcam, ab32503), rabbit monoclonal anti-Bcl-2 antibody (1:2,000, abcam, ab32124), rabbit monoclonal anti-COX-2 antibody (1:1,000, abcam, ab120295), rabbit polyclonal anti-iNOS antibody (1:1,000, abcam, ab178945), rabbit polyclonal anti-BDNF antibody (1:1,000, abcam, ab108319), rabbit polyclonal anti-NGF antibody (1:1,000, abcam, ab52987), rabbit polyclonal anti-NLRP3 antibody (1:1,000, abcam, ab263899), rabbit polyclonal anti-TLR4 antibody (1:1,000, abcam), rabbit polyclonal anti-NF-kB p65 antibody (1:1,000, abcam).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Deficiency of Orexin Receptor Type 1 in Dopaminergic Neurons Increases Novelty-Induced Locomotion and Exploration

doi: 10.1101/2023.08.06.552140

Figure Lengend Snippet: Representative images of RNAscope in situ hybridization in SN and paranigral (PN), parainterfascicular (PIF) and parabrachial (PBP) subnuclei of the ventral tegmental area (VTA), of (A) a control and (B) an Ox1R ΔDAT mouse. Amplifications of the (C) left and (D) right marked areas in white squares in (A). Amplifications of the (E) left and (F) right marked area in white squares in (B). White, tyrosine hydroxylase (Th); magenta, Ox1R; green, Ox2R. Scale bar: 100 µm (A, B) or 50 µm (C-F). Representative images of an overview in SN and VTA were shown in figure 1—figure supplement 1. Percentages of (G) Ox1R and (H) Ox2R positive neurons in Th positive neurons. IF, interfascicular subnuclei of VTA. Control, n = 3; Ox1R ΔDAT , n = 4. Data are represented as means ± SEM. **p < 0.01; ****p < 0.0001; as determined by two-way ANOVA followed by Sidak’s post hoc test. (I) Schematic illustration of Ox1R and Ox2R expression in dopaminergic neurons in the VTA and SN. 3 control mice were analyzed and N indicates the mean dopaminergic neuron numbers in the respective subregions. Magenta, dopaminergic neurons only expressing Ox1R; green, dopaminergic neurons only expressing Ox2R; yellow, dopaminergic neurons expressing both Ox1R and Ox2R; grey, dopaminergic neurons expressing neither Ox1R or Ox2R. (J-L) Effect of orexin A on dopaminergic SN neurons analyzed by Ca 2+ imaging with GCaMP6. Recordings were performed in brain slices from control and Ox1R ΔDAT male mice with GCaMP6 expressed in dopaminergic SN neurons for Ca 2+ imaging. (J) Schematic illustration of the SN. The red frame indicates the region where calcium imaging was performed. (K, L) Orexin A effect on [Ca 2+ ] i of dopaminergic SN neurons. (K) Heat maps of 5 individual dopaminergic SN neurons from control (top) and Ox1R ΔDAT mice. The recordings show the responses to 100 nM orexin A. The dashed lines indicate the range where the responses were quantified. (L) Mean Ca 2+ responses upon 100 nM orexin A application. Data are shown as the percentage of the maximal response to high K + saline. Bar graphs represent means ± SEM. The stacked bar shows the percentage of individual dopaminergic neurons in control mice in which the increase in [Ca 2+ ] i was larger than 3 x SD of the control, thus defining them as orexin A responsive (see Materials and Methods). The significance of mean changes in [Ca 2+ ] i during orexin A application were compared using one-sample t-tests. **** p < 0.0001. n values are given in brackets.

Article Snippet: Orexin A (ab120212, Abcam) in the concentration of 100 nM was applied for 8 min. To analyze the orexin effect, we compared the fluorescence measured during 4 min intervals that were recorded immediately before and at the end of the peptide application.

Techniques: In Situ Hybridization, Expressing, Imaging

Journal: bioRxiv

Article Title: Deficiency of Orexin Receptor Type 1 in Dopaminergic Neurons Increases Novelty-Induced Locomotion and Exploration

doi: 10.1101/2023.08.06.552140

Figure Lengend Snippet: (A) Total traveling distance, (B) ambulating time, (C) vertical activity and (D) stereotypic activity of male mice in the open field test. Control, n = 14; Ox1R ΔDAT , n = 15. (E) Total traveling distance, (F) ambulating time, (G) vertical activity and (H) stereotypic activity of female mice in the open field test. Control, n = 14; Ox1R ΔDAT , n = 12. Locomotor activity upon intracerebroventricular (ICV) injection of saline (NS) or orexin A in (I) male and (K) female control and Ox1R ΔDAT mice. (J, L) Quantification of the area under curve (AUC) after orexin injection to (J) male and (L) female mice. Male, n = 8; female-control, n = 9; female-Ox1R ΔDAT , n = 10. Data are represented as means ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001; as determined by unpaired two-tailed Student’s t-test (A-C, E-H), or two-way ANOVA followed by Sidak’s post hoc test (J, L).

Article Snippet: Orexin A (ab120212, Abcam) in the concentration of 100 nM was applied for 8 min. To analyze the orexin effect, we compared the fluorescence measured during 4 min intervals that were recorded immediately before and at the end of the peptide application.

Techniques: Activity Assay, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Deficiency of Orexin Receptor Type 1 in Dopaminergic Neurons Increases Novelty-Induced Locomotion and Exploration

doi: 10.1101/2023.08.06.552140

Figure Lengend Snippet: 3D maps of p-values in PET imaging studies comparing Ox1R ΔDAT and control mice, after intracerebroventricular (ICV) injection of (A) saline (NS) and (B) orexin A. Brain areas with significant changes are indicated. Control-NS, n = 8; control-orexin, n = 6; Ox1R ΔDAT , n = 8. M2, secondary motor cortex; MPA, medial preoptic area; Pir, piriform cortex; IEn, intermediate endopiriform claustrum; DEn, dorsal endopiriform claustrum; VEn, ventral endopiriform claustrum; LSS, lateral stripe of the striatum; BNST, the dorsal bed nucleus of the stria terminalis; HDB, nucleus of the horizontal limb of the diagonal band; MCPO, magnocellular preoptic nucleus; S1Sh, primary somatosensory cortex, shoulder region; S1HL, primary somatosensory cortex, hindlimb region; S1BF, primary somatosensory cortex, barrel field; S1Tr, primary somatosensory cortex, trunk region; V1, primary visual cortex; V2L, secondary visual cortex, lateral area; SubCV, subcoeruleus nucleus, ventral part; Gi, gigantocellular reticular nucleus; IRt, intermediate reticular nucleus; LPGi, lateral paragigantocellular nucleus; Sp5O, spinal trigeminal nucleus, oral part; Sp5I, spinal trigeminal nucleus, interpolar part; sp5, spinal trigeminal tract.

Article Snippet: Orexin A (ab120212, Abcam) in the concentration of 100 nM was applied for 8 min. To analyze the orexin effect, we compared the fluorescence measured during 4 min intervals that were recorded immediately before and at the end of the peptide application.

Techniques: Imaging, Injection

Journal: bioRxiv

Article Title: Deficiency of Orexin Receptor Type 1 in Dopaminergic Neurons Increases Novelty-Induced Locomotion and Exploration

doi: 10.1101/2023.08.06.552140

Figure Lengend Snippet: (A) Representative images of c-Fos and tyrosine hydroxylase (Th) staining in LPGi of control and Ox1R ΔDAT mice injected (ICV) with saline (NS) and orexin A. Quantification of (B) Th fluorescence and (C) c-Fos positive neurons in LPGi. (D) Representative images of D1 and D2 subtypes of dopamine receptor (DRD1 and DRD2) in LPGi of control and Ox1R ΔDAT mice. (E) Quantification of DRD2 fluorescence in LPGi. (F) Representative images of a negative control staining of DRD1 and DRD2 in LPGi of control mice, and (G) a positive control staining around the lateral ventricle (LV). (H) Representative images of c-Fos and Th staining in the dorsal BNST of control and Ox1R ΔDAT mice injected (ICV) with saline or orexin A. Quantification of (I) Th fluorescence and (J) c-Fos positive neurons in dorsal BNST. (K) Representative images of DRD1 and DRD2 in the dorsal BNST of control and Ox1R ΔDAT mice. Quantification of (L) DRD1 and (M) DRD2 fluorescence in dorsal BNST. Scale bar: 200 µm (D, F, G, K), 100 µm (A, H) or 50 µm (insertions in H). Control, n = 3; Ox1R ΔDAT , n = 4. Cyan, Th; red, c-Fos (A, H). Magenta, DRD1; cyan, DRD2; blue, dapi (D, F, G, K). Data are represented as means ± SEM. *p < 0.05; ***p < 0.001; as determined by unpaired two-tailed Student’s t-test (L, M), or two-way ANOVA followed by Sidak’s post hoc test (C, J).

Article Snippet: Orexin A (ab120212, Abcam) in the concentration of 100 nM was applied for 8 min. To analyze the orexin effect, we compared the fluorescence measured during 4 min intervals that were recorded immediately before and at the end of the peptide application.

Techniques: Staining, Injection, Fluorescence, Negative Control, Positive Control, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Targeting N -glycosylation of 4F2hc mediated by glycosyltransferase B3GNT3 sensitizes ferroptosis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41418-023-01188-z

Figure Lengend Snippet: A . Pipeline for identifying glycoproteins of interest shared by 211 DGPs and 387 ferroptosis-related proteins (FRPs). B Heatmap of normalized expression of differential glycopeptide across the vehicle and RSL3 groups. The upper and bottom panels show the differential N - and O -glycopeptides, respectively. The dark blue- and red bars denote vehicle and RSL3, respectively. The brown- and gray bars represent N -linked DGPs ( N -DGPs) and O -linked DGPs ( O -DGPs), respectively. C Venn diagram displaying the number of overlapping and unique proteins from 200 N -DGPs, 23 O -DGPs, and 387 FRPs. D Volcano plot showing 7 DGPs identified from N- and O -glycoproteomic. E LC–MS/MS-based analysis of the targeted differential N -linked glycosylation sites Asn365 of the 4F2hc in PANC-1 cells treated with or without 0.8 μM RSL3 for 12 h. The b11 and y4 ions represent the peptide fragmentation of Asn365 which was detected in RSL3-treated PANC-1 cells. F Western blotting analysis of the glycosylation changes in 4F2hc protein extracted from PANC-1 cells was treated with PNGase F and O -glycosidase for 1 h at 37 °C. G PANC-1, AsPC-1, and MIA PaCa-2 cells were treated with or without PNGase F for 1 h at 37 °C followed by immunoblot analysis. Purple- and cyan-triangle denote high- and low-glycosylation, respectively. H Cell lysates from six PDAC tumors treated with or without PNGase F for 1 h at 37 °C. I Flow chart presenting an investigation to screen out the putative target genes from glycosyltransferase genes (GTGs) and differentially expressed genes (DEGs) based on RNA-seq. J Left, Venn diagram displaying the number of overlapping genes from 207 GTGs and 5571 DEGs. Right, heatmap of all the up- and down-regulated hub genes intersection from 207 GTGs and 5571 DEGs across the vehicle and RSL3 groups. The brown bar represents upregulated DEGs, and the gray bar represents downregulated DEGs, the genes marked in red are four GTGs associated with PDAC prognosis. K qRT-PCR profiling of the expression of 4 selected upregulated genes involved in glycosyltransferases in PANC-1 and MIA PaCa-2 cells treated with vehicle or RSL3. L Coomassie blue stained SDS gels of affinity-purified protein complexes co-immunoprecipitated by the 4F2hc antibody from SLC3A2 overexpressed PANC-1 cells (line 3). Venn diagram displaying the 15 overlapping proteins from 1708 precipitated proteins and 185 glycosyltransferases (GTPs). M Expression of 4F2hc, xCT (CST#12691, 35 kDa), B3GNT3, DHODH, GPX4, and FSP1 in PANC-1 cells treated with RSL3 at the range of concentrations for 12 h. N Western blot analyzing 4F2hc, xCT (Abcam#175186, 55 kDa), B3GNT3, and GPX4 in human pancreatic epithelial nestin-expressing (HPNE) and PDAC cells (BxPC-3, MIA PaCa-2, PANC-1, AsPC-1). The western blot experiment was representative of two biological replicates with similar results. All data are shown as the mean ± SD. Statistical significance among the indicated groups was assessed by ANOVA analysis or unpaired Student’s t-test.

Article Snippet: All chemical compounds used in cell culture experiments included RSL3 (Selleck, S8155; Houston, USA), erastin (Selleck, S7242), imidazole ketone erastin (Selleck, S8877), Liproxstatin-1 (Selleck, S7699), ±-α-tocopherol (Selleck, S6104), UAMC-3203 (Selleck, S8792), Z-VAD-FMK (Selleck, S7023), Necrostatin-1 (Selleck, S8037), cycloheximide (Selleck, S7418), tunicamycin, and protein glycosylation inhibitor (Abcam, ab120296).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Western Blot, RNA Sequencing Assay, Quantitative RT-PCR, Staining, Affinity Purification, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Targeting N -glycosylation of 4F2hc mediated by glycosyltransferase B3GNT3 sensitizes ferroptosis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41418-023-01188-z

Figure Lengend Snippet: A Schematic plot of the 4F2hc and xCT complex and its function. B High expression of 4F2hc shows a positive correlation with resistance to ferroptosis inducers (RSL3 (Pearson’s correlation coefficient (PCC) = 0.329), ML162 (PCC = 0.348), and ML210 (PCC = 0.33)) in cancer cells. Data mined from the Cancer Therapeutics Response Portal (CTRP) ( http://www.broadinstitute.org/ctrp ). Box and whisker plots indicate the 10th to 90th percentile range, and minimum and maximum values, each value of distributions are PCC on the y-axis. C PANC-1 cells were treated with TM at different doses for different amounts of time, and the expression changes in 4F2hc and GPX4 were determined by western blotting. The purple- and cyan- triangles represent high- and low- glycosylation, respectively. D The expression and localization of transmembrane protein 4F2hc in PANC-1 cells treated with or without PNGase F are determined by the confocal image on the left and flow cytometry on the right. E mRNA levels of the indicated gene in SLC3A2 knockdown PANC-1 cells were quantified by qRT–PCR. F Measurement of 4F2hc, NRF2, xCT (CST#12691, 35 kDa), DHODH, and GPX4 protein expression levels in PANC-1 cells with or without 4F2hc knockdown. G Cell viability of sh SLC3A2 PANC-1 and MIA PaCa-2 cells treated with a range of RSL3 doses for 12 h. H Lipid ROS levels in shControl and sh SLC3A2 PANC-1 cells treated with or without 0.8 μM RSL3 for 8 h. BODIPY™ 581/591 C11 staining was used for FACS analysis in FITC channel. I Extracellular glutamate levels in shControl and sh SLC3A2 PANC-1 cells treated with or without 0.8 μM RSL3 for 12 h. J Intracellular GSH levels in shControl and sh SLC3A2 PANC-1 cells treated with or without 0.8 μM RSL3 for 12 h. K , L Cell viability in shControl and sh SLC3A2 PANC-1 and MIA PaCa-2 cells treated with or without 10 μg/ml TM or 0.8 μM RSL3 for 12 h. M Western blotting analyzing the protein expression of 4F2hc in SLC3A2 knockdown PANC-1 and MIA PaCa-2 cells transfected with wildtype (WT) 4F2hc, N365Q, and 4NQ plasmids, respectively. N , O Cell viability analysis of sh SLC3A2 PANC-1 ( N ) and sh SLC3A2 MIA PaCa-2 cells ( O ) transfected with WT, N365Q, and 4NQ plasmids, respectively, with or without 0.8 μM RSL3 treatment for 12 h. All data are shown as the mean ± SD. Statistical significance among the indicated groups was assessed by ANOVA analysis or unpaired Student’s t-test. At least three independent experiments except for B and D, 3 technical replicates for G. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: All chemical compounds used in cell culture experiments included RSL3 (Selleck, S8155; Houston, USA), erastin (Selleck, S7242), imidazole ketone erastin (Selleck, S8877), Liproxstatin-1 (Selleck, S7699), ±-α-tocopherol (Selleck, S6104), UAMC-3203 (Selleck, S8792), Z-VAD-FMK (Selleck, S7023), Necrostatin-1 (Selleck, S8037), cycloheximide (Selleck, S7418), tunicamycin, and protein glycosylation inhibitor (Abcam, ab120296).

Techniques: Expressing, Whisker Assay, Western Blot, Flow Cytometry, Quantitative RT-PCR, Staining, Transfection

Journal: Cell Death and Differentiation

Article Title: Targeting N -glycosylation of 4F2hc mediated by glycosyltransferase B3GNT3 sensitizes ferroptosis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41418-023-01188-z

Figure Lengend Snippet: A High expression of B3GNT3 shows a positive correlation with resistance to ferroptosis inducers (RSL3 (PCC = 0.19), ML162 (PCC = 0.206), and ML210 (PCC = 0.239)) in cancer cells. Data mined from the CTRP. B mRNA and protein levels of B3GNT3 in si B3GNT3 PANC-1 cells were quantified by qRT–PCR and western blotting. C Cell viability measurement of siControl and si B3GNT3 PANC-1 and MIA PaCa-2 cells treated with 0.8 μM RSL3 for 12 h. D The mRNA expression levels of B3GNT3 were detected in PANC-1, BxPC-3, and MIA PaCa-2 cells with indicated genotypes. E Cell viability measurement in Cas9 Control and B3GNT3 KO cell lines treated with a range of RSL3 doses for 12 h. F – H Measurement of extracellular glutamate ( F ), GSH ( G ), and lipid ROS ( H ) in Cas9 Control and B3GNT3 KO PANC-1 cells treated with 0.8 μM RSL3 for the indicated times. I Western blotting analyzing the protein expression of B3GNT3, 4F2hc, xCT, and GPX4 in PANC-1, BxPC-3, and MIA PaCa-2 cells with indicated genotypes. J CHX-chase analysis for PANC-1 and MIA PaCa-2 cells with the indicated genotypes. Cells were treated with 20 μM CHX at the indicated intervals, and then the expression of 4F2hc and GPX4 was measured by immunoblotting, and the intensity of 4F2hc and GPX4 was quantified and normalized to that at the 0-time point. K Western blotting analyzing the protein expression of B3GNT3 in B3GNT3 KO PANC-1 and MIA PaCa-2 cells transfected with 122-311 OE and 122-311 del plasmids, respectively. L , M Cell viability analysis of B3GNT3 knockout PANC-1 ( L ) and MIA PaCa-2 cells ( M ) transfected with 122-311 OE and 122-311 del plasmids, respectively, with or without 0.8 μM RSL3 treatment for 12 h. N The schematic diagram shows that B3GNT3 promotes the conversion of non-glycosylated (NG) 4F2hc into glycosylated (G) 4F2hc through N -glycosylation modification, then N -glycosylated 4F2hc translocate to the cell membrane to enhance the resistance of PDAC cells to ferroptosis. The western blotting experiment was representative of two biological replicates with similar results. All data are shown as the mean ± SD. Statistical significance among the indicated groups was assessed by ANOVA analysis or unpaired Student’s t-test. NS: no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: All chemical compounds used in cell culture experiments included RSL3 (Selleck, S8155; Houston, USA), erastin (Selleck, S7242), imidazole ketone erastin (Selleck, S8877), Liproxstatin-1 (Selleck, S7699), ±-α-tocopherol (Selleck, S6104), UAMC-3203 (Selleck, S8792), Z-VAD-FMK (Selleck, S7023), Necrostatin-1 (Selleck, S8037), cycloheximide (Selleck, S7418), tunicamycin, and protein glycosylation inhibitor (Abcam, ab120296).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Knock-Out, Modification

Journal: Cell Death and Differentiation

Article Title: Targeting N -glycosylation of 4F2hc mediated by glycosyltransferase B3GNT3 sensitizes ferroptosis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41418-023-01188-z

Figure Lengend Snippet: A Structure of human Erastin-bound 4F2hc-xCT complex (PDB: 7EPZ), shown as surface (left) and cartoon (middle). Detailed view of four glycosylation sites, shown as red sticks (right). Cyan denotes 4F2hc, gray denotes xCT, and orange denotes carbohydrate. B Prediction of protein stability changes upon N -glycosylation sites mutation in 4F2hc using DynaMut ( https://biosig.lab.uq.edu.au/dynamut/ ). C Prediction of the impact of glycosylation site mutations of 4F2hc on its protein flexibility and stability by PredyFlexy (one available online software for predicting root mean square fluctuations (RMSF) and β -factor based on MD simulations) ( https://www.dsimb.inserm.fr/dsimb_tools/predyflexy/ ). Red and blue lines represent the RMSF and β -factor prediction respectively deduced from flexibility class prediction based on MD simulation. The higher value of RMSF or β -factor after mutation of a glycosylation site indicates that a residue is more rigid and vice versa. D Western blot analyzing the protein expression of 4F2hc and GPX4 in sh SLC3A2 PANC-1 and MIA PaCa-2 cells reconstituted with N365Q and 4NQ mutant after treated with CHX (20 μM) at indicated intervals. E The intensity of 4F2hc and GPX4 was quantified using ImageJ software and values were normalized to that at the 0-time point for comparative analysis. F PANC-1 and MIA PaCa-2 cells treated with 20 μM CHX at the indicated intervals in the presence of 10 μg/ml tunicamycin or not. G Quantification of indicated proteins using ImageJ software and values were normalized to that at the 0-time point for comparative analysis.

Article Snippet: All chemical compounds used in cell culture experiments included RSL3 (Selleck, S8155; Houston, USA), erastin (Selleck, S7242), imidazole ketone erastin (Selleck, S8877), Liproxstatin-1 (Selleck, S7699), ±-α-tocopherol (Selleck, S6104), UAMC-3203 (Selleck, S8792), Z-VAD-FMK (Selleck, S7023), Necrostatin-1 (Selleck, S8037), cycloheximide (Selleck, S7418), tunicamycin, and protein glycosylation inhibitor (Abcam, ab120296).

Techniques: Mutagenesis, Software, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: Targeting N -glycosylation of 4F2hc mediated by glycosyltransferase B3GNT3 sensitizes ferroptosis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41418-023-01188-z

Figure Lengend Snippet: A Laser scanning confocal microscope (LSCM) images of PANC-1 and MIA PaCa-2 cells after treatment with 10 µg/ml TM for 12 h and then incubation with CHX for 0, 6, and 12 h. 4F2hc, xCT, and DAPI were stained with Alexa Fluor ® 488, Alexa Fluor ® 594, and 4′,6-diamidino-2′-phenylindole dihydrochloride, respectively. Scale bar, 20 μm. B , C Co-IP analysis of the effect of de-glycosylation mutants N365Q and 4NQ on the interaction between 4F2hc and xCT (Abcam#175186, 55 kDa) in sh SLC3A2 PANC-1 cells. D , E Co-IP analysis of the interaction between 4F2hc and xCT (Abcam#175186, 55 kDa) in B3GNT3 KO PANC-1 cells reconstituted with 122-311 OE and 122-311 del plasmids. F The confocal image determines the co-localization of transmembrane protein 4F2hc and xCT in sh SLC3A2 MIA PaCa-2 cells transfected with WT, N365Q, or 4NQ or in MIA PaCa-2 cells treated with TM. Scale bar, 10 μm. G Flow cytometry examines the membrane expression of xCT in sh SLC3A2 MIA PaCa-2 cells transfected with WT, N365Q, or 4NQ or in MIA PaCa-2 cells treated with TM.

Article Snippet: All chemical compounds used in cell culture experiments included RSL3 (Selleck, S8155; Houston, USA), erastin (Selleck, S7242), imidazole ketone erastin (Selleck, S8877), Liproxstatin-1 (Selleck, S7699), ±-α-tocopherol (Selleck, S6104), UAMC-3203 (Selleck, S8792), Z-VAD-FMK (Selleck, S7023), Necrostatin-1 (Selleck, S8037), cycloheximide (Selleck, S7418), tunicamycin, and protein glycosylation inhibitor (Abcam, ab120296).

Techniques: Microscopy, Incubation, Staining, Co-Immunoprecipitation Assay, Transfection, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Inherited retinal degeneration: T-type voltage-gated channels, Na + /Ca 2+ -exchanger and calpain-2 promote photoreceptor cell death

doi: 10.1101/2023.07.16.549200

Figure Lengend Snippet: A ) Balloon plot showing time-dependent whole-retina expression changes (post-natal day (P) 7 to P21) of cyclic nucleotide–gated channel (CNGC), Ca 2+ -release activated channel (CRAC), Na + /Ca 2+ exchanger (NCX), and voltage-gated Ca 2+ channel (VGCC). B ) Deviation plot highlighting expression changes for CNGC, CRAC, NCX, and VGCC in P13 rd1 whole retina. C ) Balloon plot showing scRNA-seq data and time-dependent expression changes (post-natal day (P) 11 to P17) of CNGC, CRAC, NCX, and VGCC in rd1 rod photoreceptors. D ) Deviation plot highlighting expression changes for CNGC, CRAC, NCX, and VGCC in P13 rd1 rod photoreceptors. E ) TUNEL assay labelling dying cells (magenta) in rd1 and wild-type ( wt ) retinal explant cultures. DAPI (grey) was used as a nuclear counterstain. Untreated (Untr.) wt and rd1 retina were compared to retina treated with 50 µM CNGC inhibitor (L-cis diltiazem), 20 µM CRAC inhibitor (CM4620), 40 µM NCX inhibitor (SN-6), 100 µM L-type VGCC inhibitor (D-cis diltiazem), 10 µM T-type VGCC inhibitor (TTA-A2), and 15 µM α 2 δ subunit VGCC ligand (DS5565). Scatter plot showing percent TUNEL-positive cells in the outer nuclear layer (ONL). The dashed line indicates the rd1 untr. situation, data points below this threshold indicate protective effects, data points above suggest destructive effects. Statistical testing: one-way ANOVA and Tukey’s multiple comparison post hoc test. Untr. wt : n = 5; Untr. rd1 : 18; L-cis rd1 : 6; CM4620 rd1 : 15; SN-6 rd1 : 9; D-cis rd1 : 12; TTA-A2 rd1 : 6; DS5565 rd1 : 6; error bars represent SD; significance levels: *** = p < 0.001; **** = p < 0.0001. INL = inner nuclear layer, GCL = ganglion cell layer; scale bar = 50 µm.

Article Snippet: Cultures were treated with 10 μM BAPTA-AM (ab120503; Abcam, Cambridge, UK), 50μM L-cis-diltiazem (ab120532; Abcam), 20 μM CM4620 (HY-101942; MedChemExpress, Sollentuna, Sweden), 40 μM SN-6 (HY-107658; MedChemExpress), 100 μM D-cis-diltiazem (ab120260; Abcam), 10 μM TTA-A2 (HY-111828; MedChemExpress), 15 μM DS5565 (HY-108006; MedChemExpress), and 1 μM NA-184 (kindly provided by Michel Baudry, Western University, Pomona, CA, USA), respectively.

Techniques: Expressing, TUNEL Assay, Comparison

Journal: bioRxiv

Article Title: Inherited retinal degeneration: T-type voltage-gated channels, Na + /Ca 2+ -exchanger and calpain-2 promote photoreceptor cell death

doi: 10.1101/2023.07.16.549200

Figure Lengend Snippet: A ) Calpain activity assay (blue) was performed on wild-type ( wt ) and rd1 retinal explant cultures. ToPro (red) was used as a nuclear counterstain. Untreated (Untr.) wt and rd1 retina were compared to retina treated with BAPTA-AM, L-cis diltiazem, CM4620, SN-6, D-cis diltiazem, TTA-AS, DS5565, and NA-184. Scatter plot showing percent calpain activity positive cells in the outer nuclear layer (ONL). Untr. wt : n = 11; Untr. rd1 : 23; BAPTA rd1 : 10; L-cis rd1 : 6; CM4620 rd1 : 5; SN-6 rd1 : 9; D-cis rd1 : 6; TTA-A2 rd1 : 7; DS5565 rd1 : 6; NA-184 rd1 : 8. B ) Activated calpain-1 (cyan) immunostaining was performed in wt and rd1 retinal explant cultures. DAPI (grey) was used as a nuclear counterstain. Untreated wt and rd1 retina were compared to retina treated with compounds as in A. Scatter plot showing percent displaying calpain-1 activation. Untr. wt : n = 7; Untr. rd1 : 17; BAPTA rd1 : 9; L-cis rd1 : 9; CM4620 rd1 : 10; SN-6 rd1 : 8; D-cis rd1 : 10; TTA-A2 rd1 : 8; DS5565 rd1 : 8; NA-184 rd1 : 9. C ) Activated calpain-2 (yellow) immunostaining was performed in rd1 and wt retinal explant cultures. DAPI (grey) was used as a nuclear counterstain. Untreated wt and rd1 retina were compared to retina treated with compounds as in A. Scatter plot showing percent ONL cells displaying calpain-2 activation. Untr. wt : 10; Untr. rd1 : 21; BAPTA rd1 : 9; L-cis rd1 : 9; CM4620 rd1 : 10; SN-6 rd1 : 7; D-cis rd1 : 10; TTA-A2 rd1 : 8; DS5565 rd1 : 6; NA-184 rd1 : 9;. Note the significant elevation of calpain activity/calpain-2 activation caused by the CNGC inhibitor L-cis diltiazem and the CRAC inhibitor CM4620. The opposite effect is observed for calpain-1 activation. Statistical testing: one-way ANOVA and Tukey’s multiple comparison post hoc test; significance levels: * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; error bars represent SD; INL = inner nuclear layer, GCL = ganglion cell layer; scale bar = 50 µm.

Article Snippet: Cultures were treated with 10 μM BAPTA-AM (ab120503; Abcam, Cambridge, UK), 50μM L-cis-diltiazem (ab120532; Abcam), 20 μM CM4620 (HY-101942; MedChemExpress, Sollentuna, Sweden), 40 μM SN-6 (HY-107658; MedChemExpress), 100 μM D-cis-diltiazem (ab120260; Abcam), 10 μM TTA-A2 (HY-111828; MedChemExpress), 15 μM DS5565 (HY-108006; MedChemExpress), and 1 μM NA-184 (kindly provided by Michel Baudry, Western University, Pomona, CA, USA), respectively.

Techniques: Activity Assay, Immunostaining, Activation Assay, Comparison