Journal: Cell Death & Disease

Article Title: Targeting EGFR-binding protein SLC7A11 enhancing antitumor immunity of T cells via inducing MHC-I antigen presentation in nasopharyngeal carcinoma

doi: 10.1038/s41419-024-07327-9

Figure Lengend Snippet: A Bubble chart of GO enrichment analysis for downregulated genes in SUNE1 cells post-SLC7A11 knockdown. B Upper: qPCR analysis of FAF2 mRNA post-SLC7A11 knockdown. Lower: the effect of knockdown SLC7A11 on the protein expression of FAF2 and MHC-I were verified by Western blot. β-actin were used as loading controls. C Upper: qPCR analysis of FAF2 mRNA following SLC7A11 overexpression. Lower: the effect of SLC7A11 overexpression on the protein expression of FAF2 and MHC-I were verified by Western blot. β-actin were used as loading controls. D Left: qPCR confirmation of FAF2 mRNA knockdown using siFAF2-2/3. Right: the effect of FAF2 knockdown on the protein expression of SLC7A11 and MHC-I were verified by Western blot. Tubulin were used as loading controls. E Immunofluorescence assessment of MHC-I in SUNE1 and 6-10B cells post-FAF2 knockdown. Scale bars, 50 μm. * P < 0.05, *** P < 0.001. The Welch one-way ANOVA test and Games-Howell test in SUNE1 cells, Kruskal–Wallis Test and Dunn’s test in 6-10B cells. Error bars, mean ± SEM . F Immunoblot assessment of SLC7A11, FAF2, and MHC-I in SUNE1 cells post-transfection: control, siSLC7A11, siFAF2, or combined siSLC7A11 and siFAF2. Tubulin were used as loading controls. G Left: Immunoblot of ERAD pathway proteins (VCP, OS9, SYVN1, HERP) in SUNE1 cells post-SLC7A11 knockdown (left), and in 5-8F cells post-SLC7A11 overexpression (right). H Left: Immunoblot of MHC-I in SUNE1 cells transfected with control or FAF2 siRNA for 36 h, then treated with cycloheximide (CHX, 100 μg/ml) for specified durations. Tubulin were used as loading controls. Right: The half-life of the MHC-I protein was calculated. I Immunoblot assessment of MHC-I in SUNE1 and 6-10B cells under control, MG132, or Chloroquine treatments. Tubulin were used as loading controls. J In 5-8F cells overexpressing SLC7A11, IP complexes of MHC-I were immunoblotted for ubiquitination (Ub) under control, VCP inhibitors (NMS-873, 10 μM, MERCK, SML1128), or ERAD inhibitors (Eeyarestatin I, 1.25 μM, MERCK, E1286) treatments. β-actin was used as loading controls.

Article Snippet: Western blotting was conducted in accordance with a previously established protocol [ ], using the following antibodies: SLC7A11 (1:1000, ab175186, Abcam, 35-55kD; 1:1000, 26864-1-AP, Proteintech Group, 55kD; 1:1000, 382036, Zenbio, 55kD), EGFR (1:2000, 26864-1-AP, Proteintech Group), p-EGFR (1:1000, R24173, ZenBioScience), Flag (1:2000, 66008-4-Ig,Proteintech Group), GR (1:5000, 66904-1-Ig, Proteintech Group), TAP1 (1:3000, 11114-1-AP, Proteintech Group), FAF2 (1:3000, 16251-1-AP, Proteintech Group), MHC-I (1:2000, 15240-1-AP, Proteintech Group), SYVN1 (1:1000, 13473-1-AP, Proteintech Group), OS-9 (1:1000, 10061-1-AP, Proteintech Group), VCP (1:2000, 10736-1-AP, Proteintech Group), HERP (1:1000, 10813-1-AP, Proteintech Group), Ubiquitin (1:1000, #3936, CST), β-actin (1:3000, AF7018, Affinity), Tubulin (1:10000, AC021, Abclonal), Histone H3 (1:2000, 68345-1-Ig, Proteintech Group).

Techniques: Knockdown, Expressing, Western Blot, Over Expression, Immunofluorescence, Transfection, Control, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

doi: 10.1038/s41467-023-40094-3

Figure Lengend Snippet: a Subclustering analysis of fibroblasts. Populations shown at the upper right are cholesteatoma fibroblasts. Populations shown at the lower left are skin fibroblasts. Cholesteatoma fibroblasts are labeled blue, and skin fibroblasts are labeled red at the lower left. Cholesteatoma fibroblasts were associated with five subclusters labeled 1, 7, 8, 10, and 11. b Trajectory mapping performed using Monocle 3. Undifferentiated and differentiated cells are labeled blue and red, respectively. The most differentiated cells were observed in cholesteatoma (labeled red). The differentiated cells were identical to cholesteatoma fibroblasts in subcluster 8. c INHBA expression in fibroblasts in UMAP. Cholesteatoma fibroblasts showed high levels of INHBA expression. The area of high INHBA expression was identical to the area in cholesteatoma fibroblasts in subcluster 8. d Violin plots of INHBA expression in each cluster. Subcluster 8 shows a high INHBA normalized value. e Immunofluorescence staining of cholesteatoma perimatrix. Colocalization of vimentin and INHBA immunofluorescence showed that fibroblasts in the perimatrix expressed INHBA. CD45 was not colocalized with activin A. Leukocytes exhibited no activin A expression. INHBA was labeled with Alexa Fluor 568, vimentin was labeled with Alexa Fluor 488, and CD45 was labeled with Alexa Fluor 647. Scale bars: 10 µm. f INHBA mRNA expression level, corrected for GAPDH mRNA expression, was significantly higher in cholesteatoma fibroblasts than in control skin fibroblasts ( n = 6). Data represent means ± SDs. g IL-1β, PGE 2 , and TNF-α promoted INHBA expression in human primary skin fibroblasts. IL-6 did not promote INHBA expression in ear pinna-derived fibroblasts ( n = 4). The exact p -value between the control and IL-1β is 0.0000003. Data represent means ± SDs. Statistical significance was determined using the ratio paired two-tailed t -test ( f ) and two-tailed unpaired t -test ( g ). Source data are provided as a Source Data file.

Article Snippet: Human primary skin fibroblasts (C-12302; PromoCell, Heidelberg, Germany) were cultured for 3 days in DMEM supplemented with 10% FBS containing recombinant murine IL-1β (1 ng/mL; 201-LB-005; R&D Systems), PGE 2 (10 µM; 165–10813; Wako), TNF-α (50 ng/mL; 210-TA-005; R&D Systems), or IL-6 (50 ng/mL; 201-IL-010; R&D Systems).

Techniques: Labeling, Expressing, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

doi: 10.1038/s41467-023-40094-3

Figure Lengend Snippet: IL-1β, PGE 2 , and TNF-α secreted from infiltrating CD45 + cells, particularly macrophages, induced activin A-expressing pathogenic fibroblasts; the activin A acted in conjunction with RANKL to promote ectopic osteoclastogenesis.

Article Snippet: Human primary skin fibroblasts (C-12302; PromoCell, Heidelberg, Germany) were cultured for 3 days in DMEM supplemented with 10% FBS containing recombinant murine IL-1β (1 ng/mL; 201-LB-005; R&D Systems), PGE 2 (10 µM; 165–10813; Wako), TNF-α (50 ng/mL; 210-TA-005; R&D Systems), or IL-6 (50 ng/mL; 201-IL-010; R&D Systems).

Techniques: Expressing

Journal: Molecular Neurobiology

Article Title: Chronically Low NMNAT2 Expression Causes Sub-lethal SARM1 Activation and Altered Response to Nicotinamide Riboside in Axons

doi: 10.1007/s12035-024-04480-2

Figure Lengend Snippet: Higher NMNAT2 to NAMPT ratio in DRG vs SCG neurons. a Pathway of NAD synthesis from precursor NAM and NAD consumption by SARM1. b Representative immunoblot of wild-type SCG and DRG extracts probed for NAMPT, NMNAT2, SARM1 and GAPDH (loading control). Cultures were collected at DIV7. c NMNAT2:NAMPT ratio (mean ± SEM; n = 3; * p < 0.05, paired t -test). d-f Quantification of NAMPT, NMNAT2 and SARM1 levels normalised to GAPDH (mean ± SEM; n = 3; ** p < 0.01 and ns (not significant) = p > 0.05, paired t -test)

Article Snippet: The following primary antibodies were used: mouse anti-SARM1 monoclonal antibody (1 in 5000, [ ]), mouse anti-NAMPT monoclonal antibody (1 in 2000, Cayman Chemical 10813), mouse anti-NMNAT2 monoclonal antibody (1 in 250, Merck WH0023057M1), mouse anti-GAPDH monoclonal antibody (1 in 2000, Abcam ab8245).

Techniques: Western Blot, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacokinetics/Pharmacodynamics of Peptide Deformylase Inhibitor GSK1322322 against Streptococcus pneumoniae , Haemophilus influenzae , and Staphylococcus aureus in Rodent Models of Infection

doi: 10.1128/AAC.01842-15

Figure Lengend Snippet: In vitro and in vivo activities of GSK1322322 against S. pneumoniae , H. influenzae , and S. aureus isolates

Article Snippet: For a 1-log 10 reduction in bacterial counts, values ranged from 3.6 to 31 (mean, 17 ± 8.2; median, 19) and 3.4 to 60.3 (mean, 19.5 ± 17.8; median, 14.4), respectively ( ). table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Organism MIC (μg/ml) R 2 for line fit (%) Stasis 1-log 10 reduction Max killing (log 10 reduction) f AUC f AUC/MIC f AUC f AUC/MIC S. pneumoniae 10127 0.25 99.9 2.0 8.1 3.6 14.4 2.8 1316009S 0.25 96.1 6.5 26.0 15.1 60.3 1.8 ATCC 10813 0.5 100 8.4 16.9 22.2 44.5 1.2 1307007S 1 95.8 16.1 16.1 20.1 20.1 2.7 ATCC 6303 1 99.8 19.8 19.8 24.8 24.8 2.8 1629 2 100 15.8 7.9 21.2 10.6 2.4 298443 2 99.9 26.5 13.2 31.0 15.5 2.3 336808 2 86 8.2 4.2 19.0 9.5 2.6 338860 2 99.5 2.9 1.5 6.7 3.4 2.8 340449 2 94.6 8.0 4.0 14.9 7.4 2.1 L11259 2 100 7.1 3.5 9.0 4.5 2.0 Mean ± SD NA d NA 11.0 ± 7.5 11.0 ± 7.9 17.0 ± 8.2 19.5 ± 17.8 2.3 ± 0.5 Median NA NA 8.2 8.1 19.0 14.4 2.4 H. influenzae 1998-100-126H 1 99.7 7.3 7.3 13.4 13.4 2.9 503-008H 1 99.8 7.1 7.1 13.9 13.9 2.7 08003H 2 99.9 14.5 7.2 16.7 8.3 2.7 H128 2 96.6 15.3 7.6 26.0 13.0 2.7 19001H 4 91.9 14.8 3.7 37.3 9.3 3.0 Mean ± SD NA NA 11.8 ± 4.2 6.6 ± 1.6 21.5 ± 10.2 11.6 ± 2.6 2.8 ± 0.1 Median NA NA 14.5 7.2 16.7 13.0 2.7 S. aureus 1312007A 0.5 96.6 3.9 7.8 9.0 17.9 1.8 1307005A a 0.5 98.9 3.8 7.6 7.7 15.5 2.0 X32601 a , b , c 1 99.8 1.6 1.6 4.3 4.3 3.2 1309006 a 1 99.7 0.2 0.2 2.3 2.3 2.9 PVL-2 a , b 2 99.2 1.9 0.9 5.5 2.8 3.4 PK-2 2 97 2.3 1.1 14.2 7.1 2.5 A-24 4 99.9 2.7 0.7 3.8 1.0 3.1 {"type":"entrez-nucleotide","attrs":{"text":"T63256","term_id":"667121","term_text":"T63256"}} T63256 a , b , c 4 99.9 2.0 0.5 7.1 1.8 3.4 Mean ± SD NA NA 2.3 ± 1.2 2.6 ± 3.2 6.7 ± 3.7 6.6 ± 6.6 2.8 ± 0.6 Median NA NA 2.1 1.0 6.3 3.5 3.0 Open in a separate window a Macrolide resistant. b Methicillin resistant. c Quinolone resistant. d NA, not applicable.

Techniques: In Vitro, In Vivo