Additional file 1 of T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration
Creators: name: Yan, Jie
name: Wang, Lan
name: Yang, Qian-Lu
name: Yang, Qian-Xi
name: He, Xinyi
name: Dong, Yujie
name: Hu, Zhulin
name: Seeliger, Mathias W.
name: Jiao, Kangwei
name: Paquet-Durand, François
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. Photoreceptor scRNA-Seq and effects of interventions targeting photoreceptor Ca2+-permeable channels. A) Differentially expressed genes (DEGs) in rd1 rod photoreceptors at post-natal day (P)13. B) DEGs in rd1 cone photoreceptors at P13. C) Effect of 10 µM BAPTA-AM and 40 µM SN-6 treatments on rd1*Cngb1-/- retinal cultures. TUNEL and the outer nuclear layer (ONL) thickness in treated retinas compared to Untr. rd1 specimens. D) Different interventions targeting Ca2+-permeable channels in wt retinal explant cultures. Scatter plots show percentage of TUNEL positive cells in ONL. Scale bars in C = 50 µM. Statistical significance was assessed using one-way ANOVA and Tukey’s multiple comparison post hoc test. Untr.: n=8 retinal explants from different animals; BAPTA: 5; L-cis: 4; CM4620: 6; SN-6: 7; D-cis: 6; TTA-A2: 3; DS5565: 4. Figure S2. Dose-response curves for BAPTA-AM, CM 4620, SN-6, TTA-A2, DS5565, and NA-184. A) Dose-response curve for BAPTA-AM in rd1 explant cultures. In the outer nuclear layer (ONL), 10, 25, and 50 µM BAPTA-AM significantly reduced calpain activity, PARP activity, and cell death as detected via the TUNEL assay. B) Different concentrations of CM4620 were tested in rd1 explant cultures. In the ONL, at concentrations of 20 µM and 60 µM, CM4620 significantly increased ONL calpain activity, PARP activity, and cell death, as assessed by the TUNEL assay. C) Dose-response for SN-6 in rd1 explant cultures. 20 µM and 40 µM SN-6 significantly reduced calpain activity, PARP activity, and cell death (TUNEL) in the ONL. B) Different concentrations of TTA-A2 were tested in rd1 explant cultures. In ONL, 10 µM TTA-A2 significantly reduced calpain activity, PARP activity, and cell death as assessed with the TUNEL assay. C) Dose-response for DS5565 in rd1 explant cultures. 15 µM DS5565 significantly reduced calpain activity, PARP activity, but not cell death (TUNEL) in the ONL. D) Dose-response for NA-184 in rd1 explant cultures. At concentrations of 1 µM and 10 µM NA-184 significantly reduced ONL calpain activity and cell death (TUNEL) but did not decrease PARP activity. Statistical significance was assessed using one-way ANOVA and Tukey’s multiple comparison post hoc test. Figure S3. Effects of interventions targeting photoreceptor Ca2+-permeable channels, bioinformatic analysis, and retinal NCX expression. A) Long-term treatment from P5 to P23, with 10 µM BAPTA and 40 µM SN-6, in rd1 explant cultures, compared to untreated (Untr.) wild-type (wt) and rd1 specimens. B) Balloon plot showing time-dependent expression changes (post-natal day (P) 11 to P17) of cyclic nucleotide-gated channel (CNGC), Ca2+-release activated channel (CRAC), Na+/Ca2+ exchanger (NCX), and voltage-gated Ca2+ channel (VGCC) in rd1 cone photoreceptors. C) Analysis of Ca2+/calmodulin-dependent protein kinase II (CaMK2) gene expression during rd1 photoreceptor degeneration. D) Immunostaining of Na+/Ca2+ exchanger (NCX) family. NCX1 was expressed in both inner and outer retina, while NCX2 and NCX3 were not detected. Occasional staining for NCX3 in retinal blood vessels relates to the use of anti-mouse secondary antibodies and (false positive) detection of IgG. Supplemental Table 1. Differentially expressed genes (DEGs) in rd1 whole retina at post-natal day (P)13. Supplemental Table 2. Differentially expressed genes (DEGs) in rd1 rod photoreceptors at post-natal day (P)13. Supplemental Table 3. Differentially expressed genes (DEGs) in rd1 cone photoreceptors at post-natal day (P)13. Supplemental Table 4. Ca2+-related GO terms in rod photoreceptors. Supplemental Table 5. Ca2+-related GO terms in cone photoreceptors. Supplemental Table 6. Quantification of TUNEL positive, dying cells in outer nuclear layer (ONL). Supplemental Table 7. Quantification of cells positive for calpain activity/activation in outer nuclear layer (ONL). Supplemental Table 8. Quantification of cells positive for PARP activity, PAR accumulation, and sirtuin activity in outer nuclear layer (ONL).