Journal: Neoplasia (New York, N.Y.)

Article Title: Targeting lysosomal protease CTSL promotes anti-tumor immunity and sensitizes HNSCC to PD-1 blockade by stabilizing PDK1 and activating Akt–PD-L1 axis

doi: 10.1016/j.neo.2025.101228

Figure Lengend Snippet: Correlation of CTSL expression with prognosis and PD-L1 regulation in HNSCC. (A, B) Kaplan–Meier curves linking CTSL expression to overall survival (OS) using TCGA-HNSCC data (A) and to disease-free survival (DFS) using UCSC Xena data (B). (C) Box plot comparing CTSL mRNA levels between tumor and normal tissues. (D) Scatter plot showing correlation between CTSL and PD-L1 (CD274) mRNA expression. (E) Western blot evaluating endogenous CTSL protein across HNSCC cell lines. (F) Western blot analysis of PD-L1 in Cal27 and SAS cells after CTSL knockdown. (G) Flow cytometry evaluating surface PD-L1 expression following CTSL knockdown. (H) Western blot assessing PD-L1 protein following CTSL overexpression. (I) Flow cytometry quantifying PD-L1 surface expression post CTSL overexpression. (J) Western blot detecting PD-L1 levels after treatment with the CTSL inhibitor Z-FY-CHO. (K) Flow cytometry assessing membrane PD-L1 expression post Z-FY-CHO treatment.

Article Snippet: The mouse HNSCC cell line (Meer, C57BL/6 background) was from iCell, and the mouse hepatoma cell line Hepa1-6 was from ATCC.

Techniques: Expressing, Western Blot, Knockdown, Flow Cytometry, Over Expression, Membrane

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: In order to generate a TRPV4 knockout C57BL/6N mouse line with the CRISPR-Cas9 genome editing system, two single-guide RNAs (sgRNA-1, 5′-CAGGTGGTCGAGTACCAGCCCGG-3′, and sgRNA-2, 5′-CTTGCATAGTAGGGTGCTAGGGG-3′) were designed (Cyagen Biosciences).

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: In order to generate a TRPV4 knockout C57BL/6N mouse line with the CRISPR-Cas9 genome editing system, two single-guide RNAs (sgRNA-1, 5′-CAGGTGGTCGAGTACCAGCCCGG-3′, and sgRNA-2, 5′-CTTGCATAGTAGGGTGCTAGGGG-3′) were designed (Cyagen Biosciences).

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: In order to generate a TRPV4 knockout C57BL/6N mouse line with the CRISPR-Cas9 genome editing system, two single-guide RNAs (sgRNA-1, 5′-CAGGTGGTCGAGTACCAGCCCGG-3′, and sgRNA-2, 5′-CTTGCATAGTAGGGTGCTAGGGG-3′) were designed (Cyagen Biosciences).

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: In order to generate a TRPV4 knockout C57BL/6N mouse line with the CRISPR-Cas9 genome editing system, two single-guide RNAs (sgRNA-1, 5′-CAGGTGGTCGAGTACCAGCCCGG-3′, and sgRNA-2, 5′-CTTGCATAGTAGGGTGCTAGGGG-3′) were designed (Cyagen Biosciences).

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: In order to generate a TRPV4 knockout C57BL/6N mouse line with the CRISPR-Cas9 genome editing system, two single-guide RNAs (sgRNA-1, 5′-CAGGTGGTCGAGTACCAGCCCGG-3′, and sgRNA-2, 5′-CTTGCATAGTAGGGTGCTAGGGG-3′) were designed (Cyagen Biosciences).

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: In order to generate a TRPV4 knockout C57BL/6N mouse line with the CRISPR-Cas9 genome editing system, two single-guide RNAs (sgRNA-1, 5′-CAGGTGGTCGAGTACCAGCCCGG-3′, and sgRNA-2, 5′-CTTGCATAGTAGGGTGCTAGGGG-3′) were designed (Cyagen Biosciences).

Techniques: Binding Assay, Ligand Binding Assay