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Figure Lengend Snippet: a) Analysis of mRNA in patch-sequenced human and mouse Pvalb neurons. The heatmap presents the mRNA levels (transcripts per million [TPM]) in individual human (n = 21) and mouse (n = 10) neurons for the genes KCNJ1–16 encoding Kir family channels, as well as the housekeeping genes GAPDH and ACTB . The abscissa presents Pvalb neurons with their subtype identity defined by their gene expression pattern in the Allen Institute Neocortical Neuron Type Identification System (Pvalb neuron type identity codes shown in the abscissa). Human and mouse cells exhibit mRNA of KCNJ3 (Kir3.1) and KCNJ6 (Kir3.2), and also KCNJ9 (Kir3.3) which is higher in mouse. In addition, the human cells express KCNJ4 (Kir.2.3). p-values for the difference between human and mouse are presented on the left (MANOVA with Bonferroni’s post hoc test) with significance difference denoted by red text. b) The heatmap presents the Allen Institute data for the mean KCNJ gene mRNA level (TPM) in the different Pvalb neuron subtypes in humans and mice. The mRNA levels are high in human and mouse cell types for KCNJ3 and KCNJ6 . Mouse cells have higher expression of KCNJ4 , KCNJ9, KCNJ11 , and KCNJ12 (MANOVA with Bonferroni’s post hoc test). c) The plot presents the lack of an association between the Kir channel gene mRNA level (TPM, summed for all KNCJ types) and input rectification ratio (r Rin ). r Rin = 1 indicates the lack of a Kir channel effect (Spearman’s correlation test).

Article Snippet: For dSTORM microscopy rabbit Anti-GIRK1 polyclonal was used (1:200, #APC-005, https://www.alomone.com/p/anti-kir3-2-girk2/APC-006 ).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Decoding subtype development and function in human pluripotent stem cell-derived midbrain dopaminergic neurons

doi: 10.64898/2026.09.23.753784

Figure Lengend Snippet: a , Experimental schematic of TGFβ pathway modulation during mDA progenitor patterning. b, PITX3::GFP reporter design (top), flow cytometry of PITX3 CRISPRa activation (middle), and immunofluorescence (IF) for PITX3 and TH at D30 (bottom). c, Flow cytometry of PITX3, CD171 and CD99 at D25. d, Flow cytometry of ALDH activity and CD99 at D25. e, Flow cytometry of PITX3 expression and ALDH activity in the indicated gated subpopulations, with quantification of the percentage of PITX3+ALDH+ cells in each subpopulation. One-way ANOVA; n = 4. f, RT–qPCR of the indicated genes in D25 control and Activin A/BMP7-patterned populations. Student’s t -test; n = 3. g, Flow cytometry of PITX3 and ALDH activity in the indicated CD99-CD56+ populations. h, IF for PITX3, ALDH1A1 and CALB1 at D26. Scale bars, 100 μm. i, Schematic illustrating mDA subtype patterning through modulation of TGF-β signaling. j, Schematic of the day 25 sorting strategy and RT–qPCR analysis of the indicated genes in sorted day 25 populations. One-way ANOVA; n = 3–4. k, IF for ALDH1A1, PITX3 and CALB1 at D26 in populations derived from D25 sorted cells (left), with quantification of relative ALDH1A1 expression (right). Scale bars, 100 μm. One-way ANOVA; n = 4–8. l, Flow cytometry of TH and ALDH activity in the indicated CD99− and CD99+ populations, with quantification of the percentage of CD99-TH+ALDH+ cells. One-way ANOVA; n = 3. m . Schematic of hPSC-A9/A10 transplantation into the mouse striatum (top); IF staining for GIRK2, TH and CALB1 in A9 and A10 grafts at 6 months post-transplantation, with quantification of GIRK2+TH+ and CALB1+TH+ cells in the grafts. Student’s t -test; n = 3. All error bars in represent s.e.m.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against GIRK2 (1:100; Alomone Labs, APC_006-GP) and calbindin (1:200; Cell Signaling Technology, 13176).

Techniques: Flow Cytometry, Activation Assay, Immunofluorescence, Activity Assay, Expressing, Quantitative RT-PCR, Control, Analysis, Derivative Assay, Transplantation Assay, Staining