Journal: MedComm

Article Title: Blocking Extracellular Cyclophilin A‐Mediated Adhesion Protects Against Bacterial Infection

doi: 10.1002/mco2.71063

Figure Lengend Snippet: eCypA is critical for the adhesion of GAS and S.P (A) C57BL/6 mice ( n = 7) were infected intranasally with PBS or 3000 PFU of influenza A/WSN/33 (H1N1) on Day 0 followed by intraperitoneal injection of isotype IgG (IgG) or anti‐CypA mAb (100 µg/mouse) on Day 1, and then infected intranasally with GAS (1.0 × 10 7 CFU), S. P (1.0 × 10 7 CFU), K. P (1.0 × 10 7 CFU), or PBS on Day 3. (B) Adherence of GAS in the lungs of C57BL/6 mice on Day 4. (C) Adherence of S. P in the lungs of C57BL/6 mice on Day 4. (D) The lung index (100× lung/body weight) was calculated on Day 4. (E) The body weights of C57BL/6 mice were monitored during Days 0–4. (F) Hematoxylin and eosin staining of mouse lungs on Day 4 (left). The severity of lung injury was analyzed in a blinded manner (right). Scale bar, 150 µm. (G) Heatmap showing the results of the qPCR analysis indicating the mRNA levels of proinflammatory cytokines in the lungs of C57BL/6 mice on Day 4. (H and I) Adherence of GAS (H) and S. P (I) in A549 cells infected with PBS or IAV (MOI = 1) followed by treatment with IgG or anti‐CypA mAb (50 nM) for 24 h and then infection of the cells with GAS (MOI = 10) for 2 h. Data are reported as the percentage of adherent bacteria relative to the input inoculum. (J) C57BL/6 mice were preinjected intraperitoneally with eCypA (40 µg/mouse) or vehicle (PBS) for 2 h, followed by intraperitoneal injection with anti‐CypA mAb (100 µg/mouse) or vehicle (PBS) on Day 0 and then infected intranasally with GAS (1.0 × 10 7 CFU), S. P (1.0 × 10 7 CFU), K. P (1.0 × 10 7 CFU), or PBS on Day 3. (K) ELISA of eCypA in BALF from mice infected with GAS. (L–N) Adherence of GAS (K), S. P (L), and K. P (M) in the lungs of C57BL/6 mice ( n = 5) on Day 4. (O–Q) Adherence assays of GAS (N), S. P (O), and K. P (P) in A549 cells pretreated with PBS, eCypA (10 nM) or eCypA + anti‐CypA mAb (10 nM eCypA + 50 nM anti‐CypA mAb, preincubated at 37°C for 1 h) for 12 h, followed by GAS, S. P , or K. P (MOI = 10) infection for 2 h. Data are shown as the percentage of adherent bacteria relative to the input inoculum. Data shown in (B–F) and (K–N) are presented as the means ± SEMs; Data shown in (H), (I), and (O–Q) are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001 and ns = not significant. Unpaired two‐tailed Student's t test for (B) and (C), and one‐way ANOVA followed by Dunnett's test for (D–F), (H), (I), and (K–Q).

Article Snippet: A549 cells (ATCC CCL185), Madin‐Darby canine kidney cells (MDCKs, ATCC CCL‐34), and HEK293T cells (human embryonic kidney cells, ATCC CR‐3216) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Injection, H&E Stain, Analysis, Bacteria, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: MedComm

Article Title: Blocking Extracellular Cyclophilin A‐Mediated Adhesion Protects Against Bacterial Infection

doi: 10.1002/mco2.71063

Figure Lengend Snippet: eCypA upregulates the expression of bacterial adhesion receptors. (A) UMAP visualization of pooled snRNA‐seq data from 37,561 cells isolated from lung tissues of mice treated with PBS, eCypA, or eCypA + anti‐CypA mAb, and then infected with GAS. (B) Volcano plots illustrating the DEGs in alveolar type 2 (AT2) cells from PBS‐, eCypA‐, and eCypA + anti‐CypA mAb‐treated mice. (C) Dot plots illustrating the expression of the indicated genes in AT2 cells from PBS‐, eCypA‐, and eCypA + anti‐CypA mAb‐treated mice. (D) C57BL/6 mice were preinjected intraperitoneally with eCypA (40 µg/mouse) or vehicle (PBS) for 2 h, followed by intraperitoneal injection of anti‐CypA mAb (100 µg/mouse) or vehicle (PBS) on Day 0. (E and F) qPCR (E) and immunoblot (F) analyses of integrin α5 and Fn expression in the lungs of C57BL/6 mice ( n = 5) on Day 3. (G) qPCR analysis of ITGA5 , FN , CD46 , PTAFR , and MUC1 mRNA levels in A549 cells treated with PBS, eCypA (10 nM), or eCypA + anti‐CypA mAb (10 nM eCypA + 50 nM anti‐CypA mAb, preincubated at 37°C for 1 h) for 12 h. (H) Immunoblot analysis of the indicated proteins in A549 cells as described in G. Data shown in (E) are presented as the means ± SEMs; Data shown in (G) are presented as the means ± SDs; * p < 0.05, ** p < 0.01, and *** p < 0.001. One‐way ANOVA followed by Dunnett's test for (E) and (G).

Article Snippet: A549 cells (ATCC CCL185), Madin‐Darby canine kidney cells (MDCKs, ATCC CCL‐34), and HEK293T cells (human embryonic kidney cells, ATCC CR‐3216) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Isolation, Infection, Injection, Western Blot, Analysis

Journal: MedComm

Article Title: Blocking Extracellular Cyclophilin A‐Mediated Adhesion Protects Against Bacterial Infection

doi: 10.1002/mco2.71063

Figure Lengend Snippet: eCypA promotes integrin/ECM adhesion receptor expression by activating TGF‐β signaling. (A) Frequency of enriched KEGG pathways in AT2 cells from the lungs of mice across different treatment groups: PBS versus eCypA and eCypA versus eCypA+anti‑CypA. (B) Venn diagram depicting the overlap in AT2 cells from the lungs of mice across different treatment groups: PBS versus eCypA and eCypA versus eCypA+anti‐CypA mAb. Numbers indicate the count of unique or shared gene sets in each segment (e.g., 271 gene sets are uniquely upregulated in eCypA versus eCypA + anti‐CypA mAb; 551 are shared upregulated sets across both comparisons). (C) Dot plots illustrating KEGG pathways enriched among upregulated genes in AT2 cells from PBS‐ versus eCypA‐treated mice and among downregulated genes from eCypA‐ versus eCypA+anti‑CypA‐treated mice. (D) Bar plots illustrating GO enrichment for DEGs in AT2 cells from the lungs of mice across different treatment groups: PBS versus eCypA and eCypA versus eCypA+anti‑CypA. (E) qPCR analysis of ITGA5 (left) and FN (right) mRNA levels in A549 cells pretreated with DMSO, PF, or SB for 2 h, followed by treatment with eCypA (10 nM) for 12 h. (F) Immunoblot analysis of the indicated proteins in A549 cells as described in E. (G) Adherence assays of GAS in A549 cells treated as described in E and infected with GAS (MOI = 10.0) for 2 h. Data are reported as the percentage of adherent bacteria relative to the input inoculum. (H) Immunoblot analysis of the indicated proteins in A549 cells treated with eCypA (10 nM) for various durations. (I) Immunoblot analysis of the indicated proteins in A549 cells treated with eCypA (10 nM), or eCypA (10 nM) + anti‐CypA mAb (50 nM) for 30 min. Data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001, and ns = not significant. One‐way ANOVA followed by Dunnett's test for (E) and (G).

Article Snippet: A549 cells (ATCC CCL185), Madin‐Darby canine kidney cells (MDCKs, ATCC CCL‐34), and HEK293T cells (human embryonic kidney cells, ATCC CR‐3216) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Analysis, Western Blot, Infection, Bacteria

Journal: MedComm

Article Title: Blocking Extracellular Cyclophilin A‐Mediated Adhesion Protects Against Bacterial Infection

doi: 10.1002/mco2.71063

Figure Lengend Snippet: Binding of eCypA to TβR2 facilitates the formation of the TGFβ1‐TGF‐β receptor 2 complex (A) qPCR analysis of ITGA5 and FN mRNA levels in A549 cells transfected with siRNAs targeting TβR2, CD147, ACE2, or MUC18 for 48 h, and then treated with 10 nM eCypA or PBS for 12 h. (B) Immunoblot analysis of the indicated proteins in immunoprecipitated samples and whole‐cell lysates (WCL) of A549 cells treated with Fc, CypA‐Fc (200 ng/mL), R55‐Fc (200 ng/mL), CypA‐Fc + anti‐CypA mAb (1 µg/mL), or R55‐Fc + anti‐CypA mAb for 2 h. (C) Interactions of CypA with TGF‐β1, TβR1, and TβR2 were detected using ELISA. BSA was used as a negative control. (D) The blocking effect of the anti‐CypA mAb on the binding of CypA to TβR2 was detected using ELISA. (E–H) Interaction of CypA with anti‐CypA mAb (E) and TβR2 (F) was detected by SPR. The competitive effect of anti‐CypA mAb on CypA‐TβR2 interaction was detected using competitive SPR assay (G). Linearities of the CypA‐TβR2 interaction were generated from the data of the sampling points shown in (H). (I) Immunoblot analysis of the indicated proteins in A549 cells treated with eCypA (10 nM) or eCypA (10 nM) + TGF‐β pAb (50 nM) for 30 min. (J) Adherence assays of GAS in A549 cells treated with eCypA (10 nM) or eCypA (10 nM) + TGF‐β pAb (50 nM) for 12 h, and then infected with GAS (MOI = 10.0) for 2 h. Data are reported as the percentage of adherent bacteria relative to the input inoculum. (K) Immunoblot analysis of the indicated proteins in immunoprecipitated samples and WCLs of A549 cells treated with TGF‐β1 (5 ng/mL), TGF‐β1 + CypA‐His (10 nM), or TGF‐β1 + CypA‐His + anti‐CypA mAb (50 nM) for 2 h. (L and M) Interactions of TGF‐β1 with TβR2 were detected by SPR in the absence (L) or presence (M) of CypA. Data are presented as the means ± SDs; ** p < 0.01, *** p < 0.001 and ns = not significant; unpaired two‐tailed Student's t test for (D); one‐way ANOVA followed by Dunnett's test for (A) and (J).

Article Snippet: A549 cells (ATCC CCL185), Madin‐Darby canine kidney cells (MDCKs, ATCC CCL‐34), and HEK293T cells (human embryonic kidney cells, ATCC CR‐3216) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Analysis, Transfection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Negative Control, Blocking Assay, SPR Assay, Generated, Sampling, Infection, Bacteria, Two Tailed Test

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: Knocking down HDAC4 inhibits glutamine metabolism in A549 cells. (A) Glutamine uptake levels in A549 cells. (B) Glutamate production levels in A549 cells. (C) α‐Ketoglutarate production levels in A549 cells. (D) Measurement of GLS activity in A549 cells. (E) DCFH‐DA staining was used to measure the level of ROS in cells. (F) A kit was used to measure the GSH/GSSG ratio. (G) Western blotting was used to detect the expression of Nrf2, HO‐1, and NQO1. ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with three independent biological replicates. Statistical analysis was conducted using Student’s t ‐test for panels (A)‒(G). GLS, glutaminase; DCFH‐DA, 2 ′ ,7 ′ dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, oxidized glutathione; Nrf2, nuclear factor erythroid 2 related factor 2; HO‐1, heme oxygenase 1; NQO1: NAD(P)H quinone dehydrogenase 1. The original, uncropped western blot images for Figure are provided in the Supporting Information.

Article Snippet: The levels of total glutathione (T‐GSH) and GSSG in A549 cells were measured using a GSH/GSSG ratio detection kit (E‐BC‐K097‐M, Elabscience, China) following the manufacturer’s instructions.

Techniques: Activity Assay, Staining, Western Blot, Expressing, Analysis

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: HDAC4 promotes SLC38A2 expression through HIF‐1α. (A) Western blot detection of SLC38A2 expression in A549 cells with HDAC4 knockdown. (B) Western blot detection of SLC38A2 expression in A549 cells with HDAC4 overexpression. (C) Western blot detection of HIF‐1α expression in A549 cells with HDAC4 knockdown. (D) Western blot detection of HIF‐1α expression in A549 cells with HDAC4 overexpression. (E) JASPAR analysis of the binding sites of HIF‐1α and the promoter of SLC38A2. (F) ChIP‐qPCR detection of the enrichment level of HIF‐1α at the SLC38A2 promoter in A549 cells. (G) A promoter‐luciferase reporter assay was used to verify the binding of HIF‐1α to the SLC38A2 promoter. (H) RT‐qPCR detection of HIF‐1α mRNA expression in A549 cells with HIF‐1α overexpression. (I) Western blot detection of HIF‐1α expression in A549 cells with HIF‐1α overexpression. (J) Western blot detection of SLC38A2 expression in A549 cells. (K) RT‐qPCR detection of HIF‐1α mRNA expression in A549 cells with HIF‐1α knockdown. (L) Western blot detection of HIF‐1α expression in A549 cells with HIF‐1α knockdown. (M) Western blot detection of SLC38A2 expression in A549 cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with three independent biological replicates. Statistical analysis was conducted using Student’s t ‐test for panels (A)–(D), (F)–(I) and (K)–(L) and one‐way ANOVA for panels (J) and (M). HIF‐1α, hypoxia‐inducible factor‐1 subunit alpha; ChIP‐qPCR, chromatin immunoprecipitation quantitative polymerase chain reaction; IgG, immunoglobulin G; OE‐HIF‐1α, overexpression HIF‐1α; sh‐HIF‐1α, short hairpin RNA targeting HIF‐1α. The original, uncropped Western blot images for Figure –D, I,J, L,M are provided in the Supporting Information.

Article Snippet: The levels of total glutathione (T‐GSH) and GSSG in A549 cells were measured using a GSH/GSSG ratio detection kit (E‐BC‐K097‐M, Elabscience, China) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Analysis, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA