Fig 1: Distinct expression profile of ChR2 in Mrgprd+ and TRPV1+ nociceptors. (A) Co-localization of ChR2-EYFP (green) with Alexa-568-conjugated IB4 (red) or CGRP (red) (top) and statistical data for overlap quantification (bottom) in lumbar DRG from Mrgprd-ChR2 mice (n = 3 animals per group). (B) Representative images of ChR2-EYFP (green) co-stained with IB4 (red) or CGRP (red) in lumbar spinal cord from Mrgprd-ChR2 mice. (C) Representative images of ChR2-EYFP (green) co-stained with β-tubulin (red) in hindpaw glabrous skin from Mrgprd-ChR2 mice. (D) Co-localization of ChR2-EYFP (green) with Alexa-568-conjugated IB4 (red) or CGRP (red) (top) and statistical data for overlap quantification (bottom) in lumbar DRG from TRPV1-ChR2 mice (n = 3 animals per group). (E) Representative images of ChR2-EYFP (green) co-stained with IB4 (red) or CGRP (red) in lumbar spinal cord from TRPV1-ChR2 mice. (F) Representative images of ChR2-EYFP (green) co-stained with β-tubulin (red) in hindpaw glabrous skin from TRPV1-ChR2 mice. Scale bar = 100 μm for DRG and spinal cord sections, scale bar = 50 μm for skin sections.
Fig 2: Distribution of ChR2V in the dorsal part of the spinal cord.A–C. Immunohistochemical localization of ChR2V with the cell-type specific markers, NF200 (A), CGRP (B) or P2X3 (C). Scale bars indicate 40 µm.
Fig 3: Disruption of Jagged-driven Notch signaling has no detectable impact on PNEC and NEB cell fate specification or maintenance.(A) qPCR analysis of Ascl1 in E18.5 controls, single and double Jag1cnull; Jag2cnull mutants showing no significant difference in expression. Graph represents mean ± SEM (n = 4 Jag1 control, n = 3 Jag1cnull; n = 4 Jag2 control; n = 4 Jag2cnull; n = 4 Jag1/Jag2 control; n = 4 Jag1cnull; Jag2cnull). Student’s t-test was used to analyze data. Side panels: representative Cgrp immunofluorescence (IF) in controls and mutants (B) IF of secretory (Scgb3a2), NEB-associated SSEA1 (CC) and Cgrp (PNEC/NEB) in control and Jag mutants. (C, D) Preserved NEB microenvironment in E18.5 Jag1cnull; Jag2cnull double mutants: IF showing NEBs (Cgrp) and NEB-associated CCs (SSEA1, N1ICD, CC10 low) in double null mutants similar to controls (Ex. inset in D). (E) qPCR analysis of Upk3a in E18.5 control and double Jag1cnull; Jag2cnull mutants showing nearly abolished Upk3a expression in mutants (n = 3 in each group). Graph: mean ± SEM; ***p<0.0005 (F) ISH of Upk3a (left panels) and double labeled with Ascl1 (immunohistochemistry, right panels): Upk3a expression predominantly in NEB-associated CCs with rare signals scattered in CC elsewhere. In double Jag1cnull; Jag2cnull mutants CCs are abolished but in the NEB microenvironment and Upk3a becomes restricted to those surrounding NEBs. Scale bar in A-D, F = 40 μm. Scale bar in F inset = 20 μm.
Fig 4: CRMP2 and phosphorylated CRMP2 expression in adult rat trigeminal ganglion (TG) neurons. (A) Micrographs of a 10-μm section of an adult TG double-immunostained with CRMP2 and βIII-tubulin (neuronal marker). A merged image (CRMP2/βIII-tubulin/DAPI [nuclear stain]) shows that many CRMP2-positive cells also express βIII-tubulin immunoreactivity. Higher magnification (inset) shows the staining pattern surrounding the ophthalmic (V1) and maxillary (V2) nerve branch regions of TG. CRMP2 colocalizes with βIII-tubulin in both somas and neuronal processes of TG neurons. (B) Micrographs of a 10-μm section of an adult TG double-immunostained with CRMP2 pS522 and βIII-tubulin. Phosphorylated CRMP2 staining is absent from the soma of TG neurons and instead appears predominantly in nerve fibers, where it colocalizes with βIII-tubulin (merge and inset). CaV2.2, N-type voltage-gated Ca2+ channel; CGRP, calcitonin gene related peptide; CRMP2, collapsin response mediator protein 2; DAPI, 4',6-diamidino-2-phenylindole.
Fig 5: MYC-driven SCLC can arise in multiple lung cell types and initially expresses ASCL1. (A) Survival of RPM mice infected with indicated cell type-specific Ad-Cre viruses. Number of mice indicated in the figure. Mantel-Cox log-rank test, (****) P < 0.0001. (B) Representative H&E histology of SCLC from RPM mice initiated with indicated Ad-Cre viruses. Scale bar, 25 µm. (C) Representative immunohistochemistry (IHC) of early in situ tumor lesions for indicated neuroendocrine (NE) or non-NE markers (top) in RPM mice infected with indicated Ad-Cre viruses (left). Arrows indicate in situ tumors. Images shown are from mice collected at approximately the following time points postinfection: CMV 43 d, CGRP 55 d, CCSP 80 d, and SPC 180 d. Scale bar, 25 µm. (D) Representative IHC of large, invasive tumors for indicated neuroendocrine (NE) or non-NE markers (top) in RPM mice infected with indicated Ad-Cre viruses (left). Images shown are from mice collected at approximately the following time points postinfection: CMV 43 d, CGRP 55 d, CCSP 80 d, and SPC 180 d. Scale bar, 25 µm. (E) H-score quantification of IHC in C and D. H-score = percentage positive cells multiplied by intensity score of 0–3 (see the Materials and Methods). Approximately 70–200 tumors from three to 10 mice per condition were quantified. Data are shown as mean ± standard deviation (SD). Mann-Whitney two-tailed t-tests, (**) P < 0.01, (****) P < 0.0001. See also Supplemental Figure S1.
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