Fig 1: Generation and validation of human iPSCs and iPSC-derived cortical neurons depleted of SQSTM1(A) Representative fluorescence images of SQSTM1 KO and unedited WT iPSCs (RBi001-A) immunostained against SQSTM1 (green) and Hoechst (blue). Scale bars, 50 μm.(B) Representative fluorescence images of cortical neurons, after 80 days of differentiation, immunostained against SQSTM1 (green), MAP2 (red), CTIP2 (green), TUBB3/TUJ1 (red), TBR1 (green), SLC17A7/VGlut1 (green), and Hoechst (blue). Scale bars, 25 μm.(C) Relative mRNA expression of markers for cortical brain regions and pan-neuronal genes. Dots indicate expression mean ± SEM of neurons generated from two independent neural differentiation.(D) Representative western blot analysis of LC3-II under normal or starvation conditions, treated with or without Baf-A1 overnight. β-Actin was used as a loading control.(E and F) Bar graphs showing the band intensity ratio of LC3-II to β-actin detected in iPSC-derived neurons cultured in full medium (E) or starvation medium (F). Data are expressed as mean ± SEM of three independent experiments. Statistical differences were tested by unpaired two-tailed t test with Welch's correction. ∗p < 0.05.
Fig 2: Effect of thioperamide on OGD-induced impaired cell viability and dendritic morphology in primary neurons. (A) Effect of thioperamide (THIO) on cell viability under normal and OGD condition in primary neurons. (B–D) Representative immunocytochemical staining of MAP-2 (red) (B) and quantitative bar graph (C, D) showing the effect of thioperamide on neuronal morphology including total dendritic length (C) and dendritic intersections (D) under OGD-induced injury. Scale bar, 50 μm. Data are presented as mean ± SEM. n = 12 for each group in (A) n = 9 for each group in (C, D). ***P < 0.001 in (A, C). ***P < 0.001 vs. the control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the OGD group in (D).
Fig 3: Effects of RvD1 on the oxidative stress and synaptic growth of neurons. The primary neurons were cultured in medium containing 50 μM Hb for 24 h, and RvD1 was added at a concentration of 25 nM 30 min before Hb stimulation. a, b The production of ROS in neurons and the statistical results for each group (n = 3). c The activity of neurons was measured by the CCK-8 method (n = 6). d, e MDA content and SOD activity (n = 3). f The synapse changes of neurons, shown as white arrows. g The number of synaptic intersections in the visual field analyzed by ImageJ (n = 5). The data were analyzed by one-way ANOVA and Tukey’s post hoc multiple comparison. **p < 0.01, ***p < 0.001, #p < 0.05, and ##p < 0.01. Bar = 50 μm for ROS staining and 40 μm for MAP2 staining. n is the number of independent cell samples
Fig 4: Neuronal differentiation effect induced by knockdown of PRMT1/Prmt1 in cancer cells or NSCs.A and B, neuronal-like differentiation phenotype by knockdown of PRMT1 (shPRMT1) in A549 cells (A), and validation of knockdown effect of PRMT1 and detection of neuronal protein expression (NEUROD1, TUBB3, MAP2) in cells using immunofluorescence (IF) (B). Cells infected with lentivirus containing empty vector (shCtrl) were used as control. C and D, neuronal-like differentiation phenotype by knockdown of PRMT1 in SW480 cells (C), and validation of knockdown effect of PRMT1 and detection of neuronal proteins in cells using IF (D). E and F, neuronal-like differentiation effect in A375 cells in response to PRMT1 knockdown (E), and validation of knockdown effect of PRMT1 and detection of neuronal markers in cells using IF (F). G, neurosphere formation of primNSCs derived from mouse ESCs in NSC serum-free medium (left) and neuronal differentiation phenotype induced by knockdown of Prmt1 in primNSCs (right). H, analysis of Prmt1 knockdown effect and detection of neural stemness markers (Sox2, Nestin, Sox1, Pax3) and neuronal proteins (Neurod1, Tubb3, Map2) in neurospheres and in cells with Prmt1 knockdown using IF. In all IF assays, nuclei were counterstained with DAPI.
Fig 5: Representative confocal images of ICC of mouse brain slices on day 7 after acute hypobaric hypoxia modelling: “Intact”, “Hypoxia”, “Hypoxia + AAV-Syn-BDNF-eGFP”,”Hypoxia + AAV-Syn-GDNF-eGFP”. Blue—DAPI, Green—NeuN, Orange—GFAP, Red—MAP2. Odd rows-scale bar–100 µm (Plan-Apochromat 10×/0.3 objective). Even rows -scale bar–20 µm Plan-NeoFluar 40×/0.75 objective. A laser scanning microscope LSM 800 (Zeiss, Oberkochen, Germany) was used for the imaging.
Supplier Page from Abcam for Anti-MAP2 antibody [EPR19691]