Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate (AS) increases pro‐inflammatory cytokines release and bacterial clearance within LPS‐tolerant macrophages (n = 4). (a) LPS increased the release of TNF‐α (a1) and IL‐6 (a2) from peritoneal macrophages (PMs) in a dose‐dependent manner. (b) Schematic diagram of the establishment of the LPS‐tolerant macrophage model. (c) The level of TNF‐α (c1) and IL‐6 (c2) in LPS‐tolerant PMs (n = 5). (d) Effect of AS (5, 10 and 20 μg·ml−1) treatment on the level of TNF‐α (d1, d2) and IL‐6 (d3, d4) in LPS‐tolerant PMs and RAW264.7 cells (n = 5). (e) Effect of AS treatment (20 μg·ml−1) on the bacterial load in LPS‐tolerant RAW264.7 cells (n = 8). (f) Effect of AS (20 μg·ml−1) treatment on the mRNA level of TNF‐α (f1, f2) and IL‐6 (f3, f4) in LPS‐tolerant THP‐1 monocytes and THP‐1 derived macrophages (n = 5). (g) Effect of AS treatment (20 μg·ml−1) on the bacterial load in LPS‐tolerant THP‐1 derived macrophages (n = 8). One‐way ANOVA followed by Tukey's post hoc test; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: The vitamin D receptor is predicted to be an interactor candidate of artesunate (AS). (a) A total of 20 underlying signal molecules were selected via the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). (b) Effect of AS on the relative mRNA levels of Vdr (n = 5). (c) Effect of AS on the protein levels of VDR (n = 5). (d) Effect of Vdr siRNA (d1) and Vdr‐KD lentiviral vector (d2) on TNF‐α levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (e) Effect of Vdr‐OE lentiviral vector on TNF‐α level in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (f1) Schematic diagram of the binding assay designed in our laboratory. (f2) Effect of VD3 on the binding of AS and VDR tracked by AS fluorophores (n = 5). AS with fluorophore 12‐(7‐oxycoumarinyl‐ethoxy) dihydroartemisinin was named AS I and AS with 12‐(‐1H‐benzo [de] isoquinoline‐1, 3(2H)‐dione‐2‐ethoxy) dihydroartemisinin was named AS II. (g) Effect of VD3 (100 nM) on TNF‐α levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). One‐way ANOVA followed by Tukey's post hoc test; ns, not significant; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Plasmid Preparation, Binding Assay

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate (AS) inhibits the nuclear translocation of VDR and modulates the transcription of its target gene Atg16l1. RAW264.7 cells were treated as described in the legend of Figure 2d. (a) Immunoblotting to observe the VDR level in the nuclear lysate. (b) Immunostaining to observe the nuclear translocation of VDR. VDR was probed using Alexa Fluor 555 (red). Representative images (bar = 5 μm) (b1). The karyoplasmic ratio of VDR was quantified from 100 cells (normalized to medium) (b2). (c) ChIP analysis for the binding of VDR to the Atg16l1 promoter. The protein–DNA complex was immunoprecipitated with anti‐VDR antibody or a negative control IgG. Representative agarose gels for the VDR‐binding region in the Atg16l1 promoter and Actb DNA in the input amplified using semiquantitative PCR. (d) The binding of VDR to the Atg16l1 promoter, normalized to Actb DNA in the input, analysed by qPCR (n = 5). (e) The protein level of ATG16L1 in LPS‐tolerant RAW264.7 cells treated with AS. (f) Change in ATG16L1 protein levels in LPS‐tolerant RAW264.7 cells (Vdr‐KD) treated with AS. (g) Change in ATG16L1 protein levels in Vdr‐OE LPS‐tolerant RAW264.7 cells treated with AS. One‐way ANOVA followed by Tukey's post hoc test; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Translocation Assay, Western Blot, Immunostaining, Binding Assay, Immunoprecipitation, Negative Control, Amplification

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate's (AS) effect is autophagy‐dependent through VDR in vitro. (a) Effect of Atg16l1 siRNA on TNF‐α (a1) and IL‐6 (a2) levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (b) Effect of bafilomycin (Baf) (10 ng·ml−1) on TNF‐α (b1) and IL‐6 (b1) levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (c) Effect of 3‐MA (5 mM) on TNF‐α (c1) and IL‐6 (c2) levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (d) Effect of 3‐MA, Ly294002 (10 μM), or Baf on the bacterial clearance in LPS‐tolerant RAW264.7 cells treated with AS (n = 8). (e1) LPS increased the protein levels of LC3B‐I, LC3B‐II and ATG5 in a dose‐dependent manner in RAW264.7 cells. (e2) The protein levels of LC3B‐I, LC3B‐II and ATG5 over time in RAW264.7 cells treated with LPS (100 ng·ml−1). The level of expression peaked at 1 h. (f) Representative image of immunofluorescence staining of LC3B in LPS‐tolerant RAW264.7 cells treated with AS (bar = 2 μm). (f1) Relative fluorescent puncta indicating LC3B aggregation were quantified from 100 cells; the number in the medium group was normalized as 1 (f2). (g) The protein levels of LC3B‐II, ATG16L1 and ATG5 in LPS‐tolerant RAW264.7 cells treated with AS. (h) Changes in LC3B‐II, ATG16L1 and ATG5 protein levels in LPS‐tolerant RAW264.7 cells (Vdr‐KD) treated with AS. (i) Changes in LC3B‐II, ATG16L1 and ATG5 protein levels in LPS‐tolerant RAW264.7 cells (Vdr‐OE) treated with AS. One‐way ANOVA followed by Tukey's post hoc test; ns, not significant; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vitro, Expressing, Immunofluorescence, Staining

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate (AS) inhibits the physical interaction between VDR and NF‐κB p65 in LPS‐tolerant macrophages. RAW264.7 cells were treated as described in the legend of Figure 2d. (a) The cytoplasm (a1) and nuclear (a2) lysate were used for an IP experiment using anti‐VDR antibodies and the associated NF‐κB p65 (p65) was detected by immunoblotting (IB). (b) Immunostaining to observe the co‐localization of p65 and VDR. p65 was probed using Alexa Fluor 488 (green). VDR was probed using Alexa Fluor 555 (red). Representative images are shown (bar = 5 μm) (b1). The co‐localization of VDR and p65 (b2) and the karyoplasmic ratio of p65 (b3) was quantified from 100 cells (normalized to medium). (c) The p65 level in the nuclear lysate was detected using elisa and WB. (d) Change in the p65 level in Vdr‐KD (d1) or Vdr‐OE (d2) LPS‐tolerant RAW264.7 cells treated with AS. (e) Change in the TNF‐α level in p65‐KD (e1) or p65‐OE (e2) LPS‐tolerant RAW264.7 cells treated with AS (n = 5). One‐way ANOVA followed by Tukey's post hoc test; ns, not significant; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Schematic of Lrrk2 domain organization indicating the position of the ROC P-loop substitution T1348N. The catalytic core is shown in black; scaffold domains are indicated in grey. ( B-B’ ) Immunoblots of endogenous Lrrk2 steady-state levels in murine RAW 264.7 macrophage cell lines (WT, Lrrk2 T1348N, and Lrrk2 KO), with β-actin as loading control ( B ), and corresponding quantification of Lrrk2 abundance normalized to WT within each independent experiment ( n = 3, B’ ). ( C-C’ ) Schematic of the protocol for differentiation of primary bone marrow-derived macrophages (BMDMs) from WT and Lrrk2 T1348N KI mice using GM-CSF (granulocyte-macrophage colony-stimulating factor; 50 ng/mL) for 7 days (representative brightfield image for illustration purposes only, C ), and immunoblots of endogenous Lrrk2 steady-state levels in BMDMs, with β-actin as loading control ( C’ ). ( D - E ) Immunoblots of Lrrk2-mediated phosphorylation of Rab10 at Thr73 (pRab10 T73) in RAW 264.7 cells ( D ) treated with vehicle (CTRL), chloroquine (CQ, 50 μM, 2 h) or the Lrrk2 inhibitor MLi-2 (0.1 μM, 90 min), and BMDMs ( E ) under vehicle (CTRL) or CQ (50 μM, 2 h). β-actin is used as loading control. ( F-I ) Representative immunofluorescence images of RAW 264.7 WT, Lrrk2 T1348N and Lrrk2 KO cells treated with vehicle or L-leucyl-L-leucine methyl ester (LLOMe; 1 mM, 30 min), showing stimulus-dependent puncta formation/recruitment of Lrrk2 (left), Rab8A (middle) and Rab10 (right) ( F ). Scale bars 10 μm. Quantification shows the number of Lrrk2-positive ( G ), Rab8A-positive ( H ) and Rab10-positive ( I ) puncta per cell under vehicle and LLOMe conditions. For each biological replicate ( n = 3), multiple fields were quantified and averaged to yield one value per replicate. Data are presented as mean ± SEM unless otherwise stated; solid dots represent independent biological replicates (mean of technical replicates, shown as open dots, within each experiment). Statistical significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical test: ( B’ ) two-tailed one-sample t test. ( G-I ) two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Derivative Assay, Phospho-proteomics, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) RT-qPCR analysis of Lrrk2 transcript levels in murine RAW 264.7 cells (WT, Lrrk2 T1348N, and Lrrk2 KO), normalized and expressed relative to WT ( n = 5 replicates). ( B-B’ ) Cycloheximide (CHX) chase in RAW 264.7 WT and Lrrk2 T1348N cells (CHX, 1 ng/μL; time points as indicated), with immunoblot detection of Lrrk2 and β-actin as loading control ( B ). Densitometric quantification shows Lrrk2/β-actin expressed as a fraction of the t = 0 signal within each genotype ( B’ ). ( C-C’ ) Immunoblots of Lrrk2 levels in RAW 264.7 WT and Lrrk2 T1348N cells following inhibition of major degradation pathways (MG132, 20 μM, 16 h for proteasome inhibition; CQ, 50 μM, 16 h for lysosomal neutralization), with β-actin as loading control ( C ). p62 and LC3B are shown as pathway response markers. Quantification of Lrrk2 abundance across treatments is expressed as fold-change relative to vehicle (CTRL) within each genotype ( n = 3 independent experiments, C’ ). ( D-D’ ) Solubility fractionation of WT and T1348N Lrrk2, assessing distribution between Triton X-100 -soluble and -insoluble fractions ( D ); quantification shows soluble Lrrk2 over total, normalized to WT within each independent experiment ( n = 3, D’ ). Data are presented as mean ± SEM; solid dots represent independent biological replicates (mean of technical replicates, shown as open dots, within each experiment). Statistical significance: ns, not significant; ** p < 0.01, **** p < 0.0001. Statistical tests: ( A, D’ ) one-sample t test; ( B’ ) two-way ANOVA with Tukey’s multiple comparisons; ( C’ ) two-way ANOVA with Šídák’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Quantitative RT-PCR, Western Blot, Control, Inhibition, Neutralization, Solubility, Fractionation

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Cellular thermal shift assay (CETSA)/thermal denaturation profiling of endogenous Lrrk2 in RAW 264.7 WT and T1348N cells across the indicated temperature range, assessed by immunoblotting. ( A’ ) Quantification of Lrrk2 band intensity from ( A ), expressed as percentage of the 4°C condition and plotted as a function of temperature. Data are presented as individual measurements and are representative of two independent experiments; curves are shown as fitted trends.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Thermal Shift Assay, Western Blot

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A-A’ ) Immunoblots of p62 levels in RAW 264.7 macrophage cell lines (WT, Lrrk2 T1348N, and Lrrk2 KO), with β-actin as loading control ( A ), and corresponding quantification relative to WT ( n = 5 independent experiments, A’ ). ( B-B’ ) Immunoblots of p62 levels in primary BMDMs from WT and Lrrk2 T1348N KI mice ( B ), with quantification relative to WT ( n = 3 independent experiments, B’ ). ( C-C’ ) Autophagic flux assessment in RAW 264.7 WT, Lrrk2 T1348N, and Lrrk2 KO cells expressing the tandem mCherry-GFP-LC3 reporter under vehicle or CQ treatment (50 µM, 16 h). Representative images show GFP, mCherry, and merged channels ( C ); quantification shows the fraction of GFP-mCherry-positive puncta normalized to total mCherry puncta per cell ( n = 3 independent experiments, C’ ). (D) Immunoblots of Lrrk2 and p62 levels in brain, lung, and kidney lysates from WT and Lrrk2 T1348N KI mice collected at 1, 6, and 12 months (pooled samples per condition), with β-actin as loading control. ( E-E’’ ) Immunoblots of individual kidney lysates from WT and T1348N mice at 1 month ( E ), 6 months ( E’ ), and 12 months, with β-actin as loading control ( E’’ ). ( F-G ) Densitometric quantification of Lrrk2 ( F ) and p62 ( G ) levels in kidneys from ( E-E’’ ), expressed relative to WT at each age ( n = 5 WT mice, n = 6 Lrrk2 T1348N mice). Data are presented as mean ± SEM; solid dots represent independent biological replicates. Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical tests: ( A’ ) one-way ANOVA with Tukey’s multiple comparisons; ( B’ ) unpaired two-tailed Student’s t test; ( C’ ) Kruskal-Wallis with Dunn’s multiple comparisons; ( F, G ) two-way ANOVA with Šídák’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Expressing, Two Tailed Test