Journal: bioRxiv

Article Title: Microglial Lag3 Drives α-Synuclein–induced Neurotoxic Activated (A1) Astrocytes and Neurodegeneration

doi: 10.64898/2026.01.06.697996

Figure Lengend Snippet: (A-C) Lag3 -/- prevents α-syn PFFs-MCM-ACM toxicity as assessed by NeuN staining. ( D-E) Lag3 antibody prevents α-syn PFFs-MCM-ACM toxicity as assessed by Hoechst and PI staining. ( F-G) Lag3 -/- prevents α-syn PFFs-MCM-ACM toxicity as assessed by Hoechst and PI staining. Data: mean ± SEM; . n=3 biologically independent primary cortical neurons. Scale bar, 20 μm. ( H-J ) Co-localization of C3 and GFAP as assessed by confocal microscopy in vivo. Scale bar, 100 μm. I, Quantification of increased C3 transcripts, which is prevented in Lag3 L/L -Cx3cr1 CreER mice injected with α-syn PFF. Data: mean ± SEM; n=5 biologically independent animals. * P < 0.05; ** P < 0.01; *** P < 0.001. ns = no significance.

Article Snippet: Lag3 L/L-YFP mice were obtained from Dr. Dario Vignali (University of Pittsburgh), and Cx3cr1 CreER knock-in/knock-out mice were purchased from the Jackson Laboratory (strain #021160).

Techniques: Staining, Confocal Microscopy, In Vivo, Injection

Journal: bioRxiv

Article Title: Microglial Lag3 Drives α-Synuclein–induced Neurotoxic Activated (A1) Astrocytes and Neurodegeneration

doi: 10.64898/2026.01.06.697996

Figure Lengend Snippet: (A) Schematic diagram of the α-syn PFF experimental design. ( B-C ) Representative immunostaining for pS129 α-syn in the Cortex, Striatum, and SNpc. Green box in the left panel of the outlines area. Scale bars, 100 μm. ( D ) Representative photomicrographs from coronal mesencephalon sections containing tyrosine hydroxylase-positive neurons in the SNpc region. n=6 biologically independent animals. Scale bar, 500 μm. Unbiased stereological counts of ( E) tyrosine hydroxylase-positive and ( F ) Nissl-positive neurons in the SNpc region. Data: mean ± SEM; n=6, biologically independent animals. unpaired two-tailed Student’s T-tests were used for statistical analyses. *** P <0.001, NS, not significant. ( G-I) Lag3 L/L -Cx3cr1 CreER rescues PFF reduction in TH and DAT levels in the ventral midbrain. ( G ) Representative immunoblots of TH, DAT, and β-actin in the ventral midbrain. ( H-I ) Quantification of TH and DAT protein levels normalized to β-actin. Data: mean ± SEM; n=3, biologically independent animals. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( J-N) , Behavioral tests after PBS or α-syn PFF stereotaxic intrastriatal injection at six months in vivo. ( J-K ) grip strength of the forelimb and four limbs. ( L ) pole test. The maximum time allowed to climb down the pole was 60 seconds. ( M ) rotarod ( N ) wire hanging test. Data: mean ± SEM; n = 6∼10 biologically independent animals. A two-way ANOVA was used for statistical analysis, followed by a Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = no significance.

Article Snippet: Lag3 L/L-YFP mice were obtained from Dr. Dario Vignali (University of Pittsburgh), and Cx3cr1 CreER knock-in/knock-out mice were purchased from the Jackson Laboratory (strain #021160).

Techniques: Immunostaining, Two Tailed Test, Western Blot, Injection, In Vivo

Journal: bioRxiv

Article Title: TRPV4 Inhibition by a Natural Product Mediates Analgesia

doi: 10.64898/2026.01.05.697673

Figure Lengend Snippet: a , The time course at +100 mV showing the effect of GSK101 on hTRPV4 expressed in HEK293 cells. b , Virtual screening workflow for the discovery of TRPV4 inhibitor. c , Chemical structure of AH001 and its glucuronide metabolite AHP. d and f , I - V curves of currents of keratinocytes inhibited by AH001 ( d ) or AHP ( f ) in the presence of 5 nM GSK101. e and g , Dose-response analysis of different concentrations of AH001 ( f) or AHP ( i) on GSK101-evoked currents of hTRPV4 at +100 mV. h and l , Cryo-EM structures of hTRPV4 in the AH001-bound ( h ) and AHP-bound states ( l ). EM densities of AH001 and AHP are represented as blue and red surfaces, respectively. i and m , The partial structure of AH001 bound to TRPV4 viewed parallel to the membrane ( i ). TRPV4 AHP structure viewed parallel to the membrane ( m ). j and n , Zoomed-in view of the AH001- and AHP-binding pocket. AH001 and AHP are represented as blue and red sticks, respectively. Residues close to AH001 or AHP are shown in stick representation. EM density for AH001 and AHP is contoured at 5σ (gray mesh). The black dashed lines represent the distances (in Å) between heavy atoms involved in hydrogen bonds. k , Representative time course and curve fitting of dose-dependent inhition of ramp current at +100 mV by AH001 on hTRPV4-T527F and hTRPV4-R746A mutants.

Article Snippet: Trpv4 knock-out mice (Trpv4 -/- ) were generated by Cyagen Biosciences (Suzhou, Jiangsu, China).

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay

Journal: bioRxiv

Article Title: TRPV4 Inhibition by a Natural Product Mediates Analgesia

doi: 10.64898/2026.01.05.697673

Figure Lengend Snippet: Selectivity evaluation of AH001 on TRPV1, TRPV3, TRPA1, and TRPM8 channels expressed in HEK293 cells. a-d , Representative time course of whole-cell TRPV1 ( a ), TRPV3 ( b ), TRPA1 ( c ) and TRPM8 ( d ) currents obtained from −100 mV to +100 mV voltage ramp in HEK293 cells perfused with AH001. e-f, Dose‒response analysis of different concentrations of AH001 ( e ) and summary of the percent of inhibition by 100 µM AH001 ( f ) on agonist-evoked currents of hTRPV1 (10 µM capsaicin), hTRPV3 (30 µM 2-APB), hTRPA1 (250 µM cinnamaldehyde) and hTRPM8 (100 µM menthol). One-way ANOVA with Tukey’s post hoc test. Data are shown as the means ± SEMs. *** P < 0.001. g , Sequence alignment of the human TRPV4, TRPV1, TRPV3, TRPA1 and TRPM8 channels. The red triangles indicate the residues at the AH001 binding pocket. T527 is important for AH001 selectivity. h , The interference of native ligand binding of a total of 44 targets by 10 μM AH001, the inhibition (or stimulation) was below 20% in all cases.

Article Snippet: Trpv4 knock-out mice (Trpv4 -/- ) were generated by Cyagen Biosciences (Suzhou, Jiangsu, China).

Techniques: Inhibition, Sequencing, Binding Assay, Ligand Binding Assay

Journal: bioRxiv

Article Title: TRPV4 Inhibition by a Natural Product Mediates Analgesia

doi: 10.64898/2026.01.05.697673

Figure Lengend Snippet: a , Superposition of the TMDs from TRPV4 AH001 (closed, yellow) and TRPV4 4α-PDD (open, pink). Relative movements of domains are indicated by red arrows. b , Side-by-side comparison of the S1-S4 helices and TRP domain rearrangements in the open and closed states. c , Comparison of coupling networks at the VSLD, TRP and S4-S5 linker in closed and open states. Dashed lines represent hydrogen bonds, salt bridges, or hydrophobic interactions. The black dashed lines represent the distances (in Å) between heavy atoms. d , Side-by-side comparison of the TRP domain and S5-S6 helices rearrangements in the open and closed states. e , Comparison of coupling networks at the S5 and S6 in closed and open states. f , Curve fitting of dose-dependent inhibition of 5 nM GSK1016790A-evoked currents at +100 mV by AH001 on various TRPV4 mutants compared to WT.

Article Snippet: Trpv4 knock-out mice (Trpv4 -/- ) were generated by Cyagen Biosciences (Suzhou, Jiangsu, China).

Techniques: Comparison, Inhibition

Journal: bioRxiv

Article Title: TRPV4 Inhibition by a Natural Product Mediates Analgesia

doi: 10.64898/2026.01.05.697673

Figure Lengend Snippet: a , Representative time course of whole-cell TRPV4 current obtained from voltage ramp (−100 mV to +100 mV every 5 seconds) in DRG neurons. Cells were held at 0 mV to inactivate voltage-gated calcium and sodium channels. b , Whole-cell recordings of peak current amplitude measured at +100 mV and −100 mV of DRG neurons. c-d , Action potential recordings from DRG neurons in control and CFA model mice, with or without AH001 treatment, and under conditions of TRPV4 knockout ( c ) or retention ( d ). e-g, Bar graph showing the effects of 20 μM AH001 on firing frequency ( e ), rheobase ( f ) and threshold potential ( g ) in nociceptive neurons, n = 30 cells in six mice, one-way ANOVA with Tukey’s post hoc test. *** P < 0.001, and **** P < 0.0001.

Article Snippet: Trpv4 knock-out mice (Trpv4 -/- ) were generated by Cyagen Biosciences (Suzhou, Jiangsu, China).

Techniques: Control, Knock-Out

Journal: bioRxiv

Article Title: TRPV4 Inhibition by a Natural Product Mediates Analgesia

doi: 10.64898/2026.01.05.697673

Figure Lengend Snippet: a , Dose-dependent analgesic effects of AH001 and indomethacin in formalin test. One-way ANOVA followed by Tukey post-tests with the vehicle-treated group, n = 8. b, Time course for analgesia by AH001 and indomethacin in Hargreaves test on day 3 after CFA-induced pain model (left). Two-way ANOVA compared with the vehicle-treated group, n = 8; Compared of normalized peek analgesic effects of AH001 and indomethacin (right). Unpaired t test, n = 8. c, Time course for analgesia by AH001 and pregabalin in cold plantar test on day 8 after PTX-induced pain model (left). Two-way ANOVA compared with the vehicle-treated group, n = 6; Compared of normalized peek analgesic effects of AH001 and pregabalin (right). Unpaired t test, n = 8. d, Time course for analgesia by AH001 and pregabalin in Von Frey test on day 7 and during days 7 to 14 post-CCI induction. Two-way ANOVA compared with the vehicle-treated group, n = 8. e, Long-term analgesic effect of AH001 by using subcutaneous implantable osmotic pump. MWT means mechanical withdrawal threshold. Two-way ANOVA compared with the vehicle-treated group, n = 3. f, Analgesic effects of AH001 in acetic acid-induced writhing test. Unpaired t test, n = 8. g , Quantification of two-chamber conditioned place aversion assay. Two-way ANOVA compared with the vehicle-treated group, n = 6. Data are shown as means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. h, Time course for analgesia by AH001 in WT mice and TRPV4 KO mice on day 3 after CFA-induced pain model. Two-way ANOVA compared with the vehicle-treated group, n = 6. i, Analgesic effects of AH001 on WT mice and TRPV4 KO mice in acetic acid-induced writhing test. Two-way ANOVA compared with the vehicle-treated group, n = 8. Data are shown as means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Trpv4 knock-out mice (Trpv4 -/- ) were generated by Cyagen Biosciences (Suzhou, Jiangsu, China).

Techniques:

Journal: bioRxiv

Article Title: TRPV4 Inhibition by a Natural Product Mediates Analgesia

doi: 10.64898/2026.01.05.697673

Figure Lengend Snippet: We propose a three-step model to elucidate the conformational transition of TRPV4 from an open to a closed state upon the binding of AH001: (1) AH001 binds within the VSLD cavity, potentially triggering a decoupling motion between the S2-S3 helices and the TRP helix, (2) the S4-S5 linker is pulled towards the TRP helix, resembling a gearbox-like motion, (3) the remodeling of the S4-S5 linker may induce decoupling between the S5 and S6 helices, ultimately resulting in a clockwise rotation of M718 and gate closure. Black arrows denote potential movements upon ligand binding.

Article Snippet: Trpv4 knock-out mice (Trpv4 -/- ) were generated by Cyagen Biosciences (Suzhou, Jiangsu, China).

Techniques: Binding Assay, Ligand Binding Assay

Journal: iScience

Article Title: A luminescence-based biosensor to measure endogenous UBE3A activity

doi: 10.1016/j.isci.2025.113684

Figure Lengend Snippet: The 45aa PSMD4 peptide interacts with UBE3A through the AZUL domain EGFP and N-terminal EGFP-tagged UBE3A constructs (human isoform 1) were expressed in HEK293T cells. Constructs: AZUL domain deleted (ΔAZUL) UBE3A, UBE3A-WT and UBE3A-ligase-dead (LD, C820A). Lysates were incubated with the biotinylated-45aa peptide and pulled down with streptavidin beads. Inputs and pellets were probed with anti-GFP antibodies.

Article Snippet: Wildtype (WT) C57BL/6J mice (#000664), Ube3a knock-out mice and UBE3A-YFP knockin mice were purchased from the Jackson Laboratory and maintained in the lab. Ube3a T503A mice were generated in the lab as previously described.

Techniques: Construct, Incubation

Journal: iScience

Article Title: A luminescence-based biosensor to measure endogenous UBE3A activity

doi: 10.1016/j.isci.2025.113684

Figure Lengend Snippet: Quantification of UBE3A activity with the Firefly-45aa biosensor (A) Localization of Firefly luciferase (Firefly-Control) and Firefly-45aa biosensor in HEK293T cells. Nuclei were labeled with DAPI and immunofluorescence staining against Lamin A/C. The biosensor was detected by immunofluorescence staining using antibodies against Firefly luciferase. Representative images show the localization of the Firefly-45aa biosensor. Scale bar = 10 μm. n represents the number of cells. (B) Firefly-45aa was co-expressed with increasing amounts of EGFP-UBE3A isoform 1 (WT-iso1), 2 (WT-iso2), or 3 (WT-iso3), followed by dual-luciferase assays 24 h after transfection. two-way ANOVA, F (2, 72) = 449.04 in plasmid amount, p < 0.0001; post hoc Tukey’s multiple comparisons test (compared to WT-iso1): ∗∗∗, p < 0.0001. n = 4. (C) Firefly-45aa or Firefly-Control was co-expressed in HEK293T cells with Renilla luciferase (internal normalization control) and either EGFP-UBE3A-WT or EGFP-UBE3A-LD(C820A). Dual-luciferase assays were performed 24 h after transfection. two-way ANOVA, F (3, 32) = 33.95 in plasmid amount, p < 0.0001; post hoc Tukey’s multiple comparisons test (compared to the 0 ng group): ∗∗∗, p < 0.0001; n.s., not significant. n = 3. (D) Sensitivity of the Firefly-45aa biosensor compared to the BAR reporter with increasing amounts of EGFP-UBE3A-WT plasmid. The Firefly-45aa biosensor or the BAR reporter was co-expressed in HEK293T cells with Renilla luciferase (internal normalization control) and EGFP-UBE3A-WT. Dual-luciferase assays were performed 48 h after transfection. two-way ANOVA, F (10, 66) = 193.1 in plasmid amount, p < 0.0001; post hoc Tukey’s multiple comparisons test (compared to the 0 ng group): ∗, p < 0.005; ∗∗∗, p < 0.0001; n.s., not significant. n = 4. In all panels, data are represented as mean ± standard error of the mean (SEM) and n represents the number of experimental repeats.

Article Snippet: Wildtype (WT) C57BL/6J mice (#000664), Ube3a knock-out mice and UBE3A-YFP knockin mice were purchased from the Jackson Laboratory and maintained in the lab. Ube3a T503A mice were generated in the lab as previously described.

Techniques: Activity Assay, Luciferase, Control, Labeling, Immunofluorescence, Staining, Transfection, Plasmid Preparation

Journal: iScience

Article Title: A luminescence-based biosensor to measure endogenous UBE3A activity

doi: 10.1016/j.isci.2025.113684

Figure Lengend Snippet: The Firefly-45aa biosensor is polyubiquitinated by UBE3A (A) Overexpression of UBE3A-WT in HEK293T cells significantly reduced protein levels of the Firefly-45aa biosensor compared to UBE3A-LD(C820A), but (B) did not alter protein levels of the Firefly control (unpaired t -test). mCherry was used as a co-transfection control for normalization. n = 3. Data are represented as mean ± SEM, and n represents the number of experimental repeats. (C) Plasmids encoding 3XHA-tagged Firefly-45aa or control (Firefly-Control) were co-expressed with EGFP-UBE3A-WT or EGFP-UBE3A-LD(C820A) as shown in the table. Total protein lysates were probed with anti-GFP and anti-HA tag antibodies. 3XHA-Firefly-Control and 3XHA-Firefly-45aa were immunoprecipitated by antibodies against the HA-tag, and immunoprecipitants were blotted with antibodies against ubiquitin and the HA-tag.

Article Snippet: Wildtype (WT) C57BL/6J mice (#000664), Ube3a knock-out mice and UBE3A-YFP knockin mice were purchased from the Jackson Laboratory and maintained in the lab. Ube3a T503A mice were generated in the lab as previously described.

Techniques: Over Expression, Control, Cotransfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: iScience

Article Title: A luminescence-based biosensor to measure endogenous UBE3A activity

doi: 10.1016/j.isci.2025.113684

Figure Lengend Snippet: The Firefly-45aa biosensor reveals a spectrum of UBE3A LOF- and GOF-mutations (A) Volcano plot separating LOF (left) from GOF (right) mutations. EGFP-UBE3A-variant plasmids (all isoform 1) were transfected at the lowest amount (1.56 ng) in HEK293T cells along with the Firefly-45aa biosensor. The position of the dashed line is equivalent to adj. p = 0.05. (B) UBE3A variants resolved by principal component analysis (PCA). PCA was performed using levels of overexpressed UBE3A (measured as EGFP intensity) and the activity of Firefly-45aa from all co-transfection groups. Relative UBE3A activity and protein levels represent results from the 25 ng co-transfection group.

Article Snippet: Wildtype (WT) C57BL/6J mice (#000664), Ube3a knock-out mice and UBE3A-YFP knockin mice were purchased from the Jackson Laboratory and maintained in the lab. Ube3a T503A mice were generated in the lab as previously described.

Techniques: Variant Assay, Transfection, Activity Assay, Cotransfection

Journal: iScience

Article Title: A luminescence-based biosensor to measure endogenous UBE3A activity

doi: 10.1016/j.isci.2025.113684

Figure Lengend Snippet: UBE3A GOF activity of each isoform and LOF dominant-negative activity (A) Isoform 1, (B) isoform 2 and (C) isoform 3 EGFP-tagged UBE3A constructs (WT, G738E and G738R) were evaluated with the Firefly-45aa biosensor in a dual-luciferase assay. For isoform 1: two-way ANOVA, F (5, 54) = 825.7 in plasmid amount, p < 0.0001; For isoform 2: two-way ANOVA, F (5, 54) = 375.2 in plasmid amount, p < 0.0001; for isoform 3: two-way ANOVA, F (5, 54) = 397.5, p < 0.0001. post hoc Tukey’s multiple comparisons test (compared to WT): ∗/ # / Δ , p < 0.01, ∗∗/ ## / Δ Δ , p < 0.001, ∗∗∗/ ### / Δ Δ Δ , p < 0.0005, ∗∗∗∗/ #### / Δ Δ Δ Δ , p < 0.0001. ∗, G738E compared to WT; Δ , G738R compared to WT; # , G738E compared to G738R. n = 4. (D and E) Expression of UBE3A LOF constructs interferes WT UBE3A activity as measured with the Firefly-45aa biosensor. (D) HEK293T cells were transfected with the EGFP-UBE3A-WT plasmid at 3.125 ng/well and indicated amount (in multiples of 3.125 ng) of plasmid encoding EGFP-UBE3A-LD(C820A) and E550L. The total amount of plasmid transfected per well was held constant by adding a proportionate amount of empty vector. Statistics relative to WT condition: LD, one-way ANOVA, F (7, 24) = 76.90, p < 0.0001, n = 3; E550L, one-way ANOVA, F (7, 24) = 46.13, p < 0.0001, n = 3; post hoc Dunnett’s multiple comparisons: ∗, p < 0.05; ∗∗, p < 0.005, ∗∗∗, p < 0.0001. n = 4. (E) HEK293T cells were transfected with EGFP empty vector, EGFP-E550L or EGFP-LD(C820A) plasmid at 12.5 ng/well and the indicated amount of mCherry-UBE3A-WT (isoform1) plasmid. two-way ANOVA, F (7, 96) = 405.84, p < 0.0001, n = 4; post hoc Dunnett’s multiple comparisons to the EGFP group, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005; ∗∗∗∗, p < 0.0001; n.s., not significant. n = 4. In all panels, data are represented as mean ± SEM, and n represents experimental repeats.

Article Snippet: Wildtype (WT) C57BL/6J mice (#000664), Ube3a knock-out mice and UBE3A-YFP knockin mice were purchased from the Jackson Laboratory and maintained in the lab. Ube3a T503A mice were generated in the lab as previously described.

Techniques: Activity Assay, Dominant Negative Mutation, Construct, Luciferase, Plasmid Preparation, Expressing, Transfection

Journal: iScience

Article Title: A luminescence-based biosensor to measure endogenous UBE3A activity

doi: 10.1016/j.isci.2025.113684

Figure Lengend Snippet: Quantification of endogenous UBE3A activity with the AAV2-Firefly-45aa biosensor (A) AAV2-Firefly-Control and Firefly-45aa bicistronic vector diagrams. Expression of the Firefly-Control or the Firefly-45aa biosensor and Renilla luciferase (normalization control) was driven by the same promoter, and the translation of Renilla luciferase was mediated by an internal ribosomal entry site (IRES). AAV vectors with an EF1α promoter (pro) were generated for ubiquitous expression, or an hSyn1 promoter for neuron-specific expression. (B) Validation of the bicistronic AAV2-Firefly-45aa-IRS- Renilla and control vectors in HEK293T cells, transfected as plasmids. For Firefly-control: One-way ANOVA with post hoc Tukey’s multiple comparisons test, F (2, 18) = 3.503, p = 0.0519; n.s., not significant; n = 7. For Firefly-45aa: One-way ANOVA with post hoc Tukey’s multiple comparisons test, F (2, 18) = 521.6, p < 0.0001. n = 7. (C) Down regulation of UBE3A protein levels by ASOs against UBE3A. Total protein lysates were extracted 4 days after ASO treatment to evaluate UBE3A protein levels. Relative UBE3A levels were measured using β-actin as the normalization control and are listed below the representative western blot of UBE3A. n = 2. (D) Firefly-45aa activity in neurons treated with UBE3A ASOs at different concentrations. One day after ASO treatment, neurons were transduced with AAV2- Ef1α -Firefly-45aa-IRES-Renilla viral particles and Firefly-45aa activity was measured on DIV7 and quantified by normalization to cells untreated with ASOs (Ctrl). One-way ANOVA, F (5, 24) = 5.070, p = 0.0026, n = 5; post hoc Dunnett’s multiple comparisons test, ∗, p < 0.05; ∗∗, p < 0.005. (E) Reduced Firefly-45aa activity in Ube3a T503A (GOF mutation) neurons using the EF1α promoter-driven AAV vector relative to control AAV vector (two-way ANOVA, F (1, 10) = 7.756, p = 0.0193; post hoc Šídák’s multiple comparisons test; WT, n = 5; mT503A/p+, n = 3). (F and G) Reduced activity of Firefly-45aa driven by hSyn1 promoter in neurons from (F) Ube3a mT503A/p+ mice (unpaired t-test, WT, n = 5; mT503A/p+, n = 3) and (G) Ube3a UBEAOE2+ mice (unpaired t-test, WT, n = 8; UBEAOE2+, n = 7). (H and I) Elevated Firefly-45aa activity driven by the hSyn1 promoter in cultured neurons from (H) AS model ( Ube3a m-/p ) mice (unpaired t-test, WT, n = 8; m-/p+, n = 11), and (I) Ube3a-YFP ( Ube3a Ube3a-YFP/p+ ) mice (unpaired t-test, WT, n = 10; mUbe3a-YFP/p+, n = 11). (J) Reduced Firefly-45aa activity, but not the Firefly control, in fibroblast cells from Ube3a T503A mice relative to WT control (two-way ANOVA, F (1, 10) = 7.756, p = 0.0193; post hoc Šídák’s multiple comparisons test; WT, n = 5; mT503A/p+, n = 3). (K and L) Elevated Firefly-45aa activity in cultured fibroblast cells from (K) AS model mice (unpaired t-test, WT, n = 5; m-/p+, n = 7) and (L) Ube3a-YFP mice (unpaired t-test, WT, n = 4; mUbe3a-YFP/p+, n = 4). For panels (B and D), n represents the number of experimental repeats; for panels (E–L), n represents the number of animals. All data are represented as mean ± SEM.

Article Snippet: Wildtype (WT) C57BL/6J mice (#000664), Ube3a knock-out mice and UBE3A-YFP knockin mice were purchased from the Jackson Laboratory and maintained in the lab. Ube3a T503A mice were generated in the lab as previously described.

Techniques: Activity Assay, Control, Plasmid Preparation, Expressing, Luciferase, Generated, Biomarker Discovery, Transfection, Western Blot, Transduction, Mutagenesis, Cell Culture

Journal: bioRxiv

Article Title: Distinct Programs Drive Organ Development and Regeneration

doi: 10.1101/2025.11.20.689429

Figure Lengend Snippet: ( A ) Differentially expressed genes by intermediate state cells (IMSCs) when compared with basal cells (BCs) and luminal cells (LCs). ( B ) Gene ontology (GO) enrichment analysis of the upregulated genes in IMSCs, emphasizing pathways linked to rapid proliferation, chromosome remodeling, and differentiation. ( C ) Heatmap showing the SCENIC transcription factor activity analysis scores for the top upregulated transcription factors in IMSCs. Pou2f3 was identified as a candidate regulator of luminal differentiation during regeneration. ( D, E ) Pou2f3 expression in single cells of the UMAP plots from the BaLu (D) and Ba (E) samples. Note that Pou2f3 expressing cells overlap well with IMSCs in the Ba samples. ( F-J ) Functional validation of Pou2f3 in mammary gland regeneration. (F) qPCR analysis showing the knockdown efficiency of Pou2f3 using shRNA. N=3. ( G , H ) Green-fluorescent images showing the epithelial outgrowths of the control shScr-infected ( G ) and Pou2f3 -knockdown sh Pou2f3 -infected ( H ) whole-mount glands. Inset shows that sh Pou2f3 knockdown cells formed aggregates but failed to undergo epithelial branching as in controls. N=5. ( I , J ) Immunofluorescence staining showing reduced luminal cell differentiation in Pou2f3 -deficient cells. Note that, contrary to shScr-samples, in which K14 + (red) basal and K8 + (white) luminal cells separated and formed a lumen, no mature luminal cells expressing only K8 (encircled by a green-dotted line) were found in the sh Pou2f3 samples, and the mutant aggregate did not form a lumen (encircled by a white-dotted line). Data are mean ± SD. Statistical analysis was performed using unpaired Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.

Article Snippet: Pou2f3 knock-out mice were purchased from Cyagen Biosciences.

Techniques: Activity Assay, Expressing, Functional Assay, Biomarker Discovery, Knockdown, shRNA, Control, Infection, Immunofluorescence, Staining, Cell Differentiation, Mutagenesis

Journal: bioRxiv

Article Title: Distinct Programs Drive Organ Development and Regeneration

doi: 10.1101/2025.11.20.689429

Figure Lengend Snippet: ( A - E ) Characterization of mammary gland differentiation in vitro using a minigland culture assay. Control ( Pou2f3 -/+ ; A - A’’’ ) or Pou2f3 null ( B - B’’’ ) basal cells were embedded and cultured for 14 days. Representative images show the progression from the organoid (day 1), to branching organoid (day 4), elongating organoid (day 10), and minigland (day 14) stages. The effect of Pou2f3 loss on luminal differentiation was quantified using FACS ( C - E ). N ≥ 6. ( F - G’’ ) Immunofluorescent confocal microscopy of K14 (red) (F, G), K8 (green) (F’, G’), and merged (F’’, G’’) images of the miniglands derived from control (F-F’’) or Pou2f3 null (G-G’’) basal cells. Asterisks indicate K14 and K8 double positive cells. N = 3. ( H ) GSEA analysis of basal and luminal programs in the miniglands derived from control and Pou2f3 null basal cells. ( I ) Genomic distribution of POU2F3-binding sites in mammary epithelial cells based on CUT&Tag analysis. Note that binding sites were predominantly located in distal regulatory regions and intronic elements, suggesting a role in enhancer-mediated gene regulation. ( J ) Enrichment of POU2F3 binding regions of genes of basal and luminal marker genes based on CUT&Tag assays. ( K ) Bar chart showing the number of upregulated and downregulated basal genes bound by POU2F3. ( L ) Violin plot comparing the openness score of the chromatin of basal genes between control and null cells. Data are mean ± SD. Statistical analysis was performed using unpaired Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.

Article Snippet: Pou2f3 knock-out mice were purchased from Cyagen Biosciences.

Techniques: In Vitro, Control, Cell Culture, Confocal Microscopy, Derivative Assay, Binding Assay, Marker

Journal: bioRxiv

Article Title: Distinct Programs Drive Organ Development and Regeneration

doi: 10.1101/2025.11.20.689429

Figure Lengend Snippet: ( A-D ) Mammary gland development in Pou2f3 knockout and control mice. ( A, B ) Whole-mount carmine staining of mammary glands showing comparable branching morphology between control ( A ) and knockout ( B ) mice. ( C, D ) Quantification of ductal elongation ( C ) and branching points ( D ). Scale bars: 2 mm. N ≥ 3 mice. ( E-I ) Immunofluorescence staining of sections of the mammary epithelium using the indicated basal ( E , F ) and luminal ( E’ , F’ , G-H’ ) cell markers from Pou2f3 control ( E, G ) and knockout ( F, H ) mice. ( I ) Quantification of the percentage of ER+ luminal cells in the mammary epithelium based on FACS analysis. Scale bars: 20 µm. N ≥ 3 mice. ( J-L ) Single-cell RNA sequencing (scRNA-seq) analysis of mammary glands from Pou2f3 knockout and control mice. ( J, K ) tSNE plots showing the distribution of cell types ( J ) and samples ( K ) of single cells from Pou2f3 KO and control mice. The three circles with green dashed-lines highlight the non-overlapping, distinct clusters of cells that were supposed to be the same subtypes. ( L ) Percentage distributions of different cell subtypes in the mammary epithelium. Data are mean ± SD. Statistical analysis was performed using unpaired Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.

Article Snippet: Pou2f3 knock-out mice were purchased from Cyagen Biosciences.

Techniques: Knock-Out, Control, Staining, Immunofluorescence, RNA Sequencing

Journal: bioRxiv

Article Title: Distinct Programs Drive Organ Development and Regeneration

doi: 10.1101/2025.11.20.689429

Figure Lengend Snippet: ( A ) GSEA analysis of the proliferation-related pathways in luminal cells of the Pou2f3 mutant mammary gland when compared with control mammary glands. ( B ) KEGG analysis of the downregulated genes in Pou2f3 null basal cells, emphasizing the TNF signaling pathway and its downstream NFκB signaling pathway. ( C ) GSEA analysis of the NFκB signaling pathway genes in basal cells of the Pou2f3 mutant mammary gland when compared with control mammary glands. ( D ) Cell-cell interaction analysis using CellChat, revealing reduced TNF signaling from luminal-to-basal cells in Pou2f3 knockout mice compared to control. ( E ) Relative Tnf mRNA expression as revealed by qPCR reaction in luminal cells from Pou2f3 knockout and control mice. N = 4. ( F , G ) TNF protein expression based on FACS analysis in luminal cells of the Pou2f3 control and mutant mammary glands ( F ) and quantification of TNF fluorescent intensity ( G ). N = 4. ( H-J ) Co-culture of wild-type basal cells (red, derived from the mTmG mammary epithelium) with luminal cells derived from either Pou2f3 control ( H ) or mutant ( I ) mammary epithelium. Basal and luminal cells were mixed at a 1:1 ratio and cultured. Luminal cells were detected using K8 immunofluorescence (green) at the end of the experiment. ( J ) Quantification of the percentage of luminal cells that were derived from basal cells. Note arrowheads denote select basal-derived luminal cells (both red- and green-colored). N ≥ 12. ( K ) Relative Pou2f3 mRNA expression, as detected by qPCR, in basal cells cultured in medium with the indicated TNF protein concentration. N = 3. ( L ) Relative luciferase activity in HC11 cells carrying a construct expressing a luciferase under the control of the Pou2f3 promoter, following TNF stimulation. N = 4. ( M, N ) Relative Pou2f3 mRNA expression, as detected by qPCR, in HC11 cells cultured in medium with or without TNF protein and the indicated inhibitor. ( M ) Effect of the transcription inhibitor actinomycin D. ( N ) Effect of the MAPK inhibitors U0126. N = 3. ( O ) Model diagram of the mutual regulation of POU2F3 and TNF during mammary gland development and regeneration. Data are mean ± SD. Statistical analysis was performed using unpaired Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.

Article Snippet: Pou2f3 knock-out mice were purchased from Cyagen Biosciences.

Techniques: Mutagenesis, Control, Knock-Out, Expressing, Co-Culture Assay, Derivative Assay, Cell Culture, Immunofluorescence, Protein Concentration, Luciferase, Activity Assay, Construct

Journal: bioRxiv

Article Title: Distinct Programs Drive Organ Development and Regeneration

doi: 10.1101/2025.11.20.689429

Figure Lengend Snippet: ( A ) Protocol diagram for prostatitis induction. ( B-F ) Assessment of the percentage of luminal cells, based on FACS analysis ( B-E ) in the Pou2f3 control and null prostate under normal ( B, C ) or a prostatitis ( E, E ) condition. ( F ) Quantification of the percentage of luminal cells based on FACS. N ≥ 4 mice. ( G ) Protocol diagram for pancreatitis induction. ( H-L ) The percentage of AMY2A + cells, based immunofluorescent microscopy ( H-K’ ) in the Pou2f3 control and null pancreas under normal ( H-I’ ) or a pancreatitis ( J-K’ ) condition. ( L ) Quantification of the percentage of luminal cells based on immunofluorescent microscopy. N ≥ 4 mice. ( M, N ) H&E staining of a paraffin section of the Pou2f3 control and null pancreas after recovering from a pancreatitis condition. N ≥ 4 mice. ( O ) Diagram of a revised model of mammary gland stem cell differentiation during development and regeneration. Abbreviations: MSCs, mammary stem cells; MPCs, mammary progenitor cells; BCs, basal cells; HRLCs, hormone-responsive luminal cells; SLCs, secretory luminal cells. Data are mean ± SD. Statistical analysis was performed using unpaired Student’s t-test. **p < 0.01; ****p < 0.0001; n.s., not significant.

Article Snippet: Pou2f3 knock-out mice were purchased from Cyagen Biosciences.

Techniques: Control, Microscopy, Staining, Paraffin Section, Cell Differentiation