EMBO/soda-nel huggingface dataset:
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 4

Figure. 4. Identification of gene targets and cellular pathways functionally associated with the miR-143/-145 cluster-mediated undifferentiated/mesenchymal-like melanoma cell phenotype. (A-C) M238P cells were transfected separately with miR-143-3p, miR-145-5p or a negative control (miR-neg) mimics and RNA content was analyzed using whole genome microarrays (dataset 1, n = 2). (A) Heatmap showing the genes differentially expressed after individual miRNA mimic overexpression. (B) Overrepresentation of miRNA predicted targets in the set of downregulated transcripts following miR-143-3p and miR-145-5p mimics transfection using miRonTop webtool. Each arrow indicates the corresponding overexpressed miRNA. (C) Venn diagram showing the selection of the best target candidates (red boxes) using miR-143-3p and miR-145-5p mimics transfection as well as comparison of M238R and M238P transcriptomic profiles. (D-E) M238P cells were transduced with a miR-143/-145 construct and selected for stable expression of the cluster or transduced with a control vector, followed by RNA-seq analysis (dataset 2, n = 2). (D) Heatmap highlighting the common predicted upstream regulators altered in cells stably expressing the miR-143/-145 cluster and M238R cells compared to control M238P cells. A subset of common regulators (out of the top 50 scores) corresponding to transcription factors (TFs), cytokines and growth factors (GFs), transmembrane receptors, kinases and phosphatases is shown. Red arrows indicate annotations related to the TGF-β pathway. (E) Venn diagram summarizing the comparison of the best-predicted targets following the 2 gain-of-function approaches. Subsets of miR-143-3p and miR-145-5p predicted targets downregulated by both mimics and stable lentivirus expression are shown (red boxes). (F) Luciferase assay in HEK cells overexpressing miR-143 or miR-145 transfected with a plasmid harboring the WT or muted sequence of the miR-143 and miR-145 binding sites present in FSCN1 3'UTR. Each bar represents the mean ± SE of experiments performed at least in triplicate. ***P≤0.001, ****P≤0.0001. P-values were calculated using Paired Student t-test. (G) RT-qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ** P≤0.01, ***P≤0.001. (H) Western Blot analysis of FSCN1 expression in M238P cells transfected with the indicated mimics.
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 5

Figure. 5. FSCN1 is a functional miR-143/-145 target contributing to the phenotypic switch towards the undifferentiated/mesenchymal-like state. (A) qPCR analysis of FSCN1, miR-143 and miR-145 expression in a 1205Lu xenograft nude mice model treated with the BRAFi Vemurafenib compared to control mice (n = 6). Paired Student t-test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. (B) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (C-E) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (C) Western Blot analysis of cell cycle markers on cell lysates from M238P cells cultured for 72 h following transfection with the indicated siRNAs. (D) Migration assay performed in Boyden chambers. Representative images showing migration of M238P cells treated with the indicated siRNAs. The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. *P≤0.05, ***P≤0.001 (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs. (F-H) BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (F) Effect of FSCN1 overexpression on cell proliferation assessed by time-lapse analysis using the IncuCyte system. Graph shows quantification of cell confluence. Two-way ANOVA analysis was used for statistical analysis. ****P≤0.0001. (G) Western Blot analysis of FSCN1, phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells. (H) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 6

Figure. 6. Regulation of actin cytoskeleton dynamics and focal adhesions by miR-143/-145 cluster /FSCN1 axis. (A, C) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (B, D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (A-B) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (C-D) Images and quantification of focal adhesions number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions (FA) number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test has been used for statistical analysis. *P≤0.01, ***P≤0.001, ****P. ≤0.0001. Scale bar 40 μm. (E-F) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (F) Images and quantification of focal adhesions (FA) number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 7

Fig. 7. Regulation of mechanosensitive transcriptional coactivators YAP and MRTF by the miR-143/-145 cluster / FSCN1 axis. (A, B, E) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). Effect of miR-143-3p or miR-145-5p overexpression on YAP (A) and MRTFA (B) nuclear translocation by immunofluorescence. Data are represented as scatter plots with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (C-D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or with a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). Effect of BRAFi or BRAFi plus MEKi on YAP (C) and MRTFA (D) nuclear translocation by immunofluorescence. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm. (E) Effect of miR-143-3p or miR-145-5p overexpression on the expression of YAP/MRTF target genes assessed by RT-qPCR. Data are normalized to the expression in control cells. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ****P≤0.0001. (F-H) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). Effect of FSCN1 downregulation on MRTFA (F) and YAP1 (G) nuclear translocation assessed by immunofluorescence in M238P. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm. (H) RT-qPCR analysis for the expression of MRTFA/YAP target genes in M238P cells transfected with the indicated siRNAs. Data are normalized to the expression in parental cells. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. Scale bar 40 μm.
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 1

Evaluation of phagocytosis‐independent stimuli on the induction of the autophagic process in human neutrophils. (A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×). (B) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively. (C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown. (D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown. (E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 2

ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 3

Effect of TLR activation on the induction of the autophagic machinery. (A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)‐treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05, n.s.=not significant. (B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown. (C) LC3B‐II/LC3B‐I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium‐treated control cells. Dotted line represents LC3B mRNA levels of medium‐treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05. (E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium‐treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05.
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 4

Impact of IL‐1β stimulation on the activation of the autophagic machinery in human neutrophils. (A) (I) Formation of acidified autophagosomes after stimulation with 10 ng/mL of IL‐1β, compared with medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. IL‐1β‐treated cells (bar 2: 65.17±10.23%) compared with control conditions (bar 1: 15.83±6.08%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05. (B) LC3B conversion in neutrophils treated with 10 ng/mL (lane II) and 100 ng/mL (lane III) of IL‐1β. (lane I: medium‐treated control cells). One representative out of five independent experiments is shown.
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 5

Implication of phagocytosis of opsonized E. coli in the activation of the autophagic process in human neutrophils. (A) Effect of 3‐MA on neutrophil phagocytic capacity of opsonized E. coli as observed by flow cytometry. Percentage of phagocytosing neutrophils after 3‐MA pretreatment (III) compared with cells incubated with opsonized bacteria (II). Data are representative of six independent experiments and are presented as mean±SD, (I: control unstimulated group), (IV) MFI representation (bar 1: control: 1±1.9, bar 2: E. coli: 387.7±33.3, bar 3: E. coli+3‐MA: 25.13±18, Wilcoxon matched‐pairs test, *p<0.05; bar 2 compared with bar 1 and bar 3 compared to bar 2). (B) (I) Presence of acidified structures in E. coli‐phagocytosing neutrophils and medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli‐phagocytosing cells (bar 2: 79.33±7.15%) compared with control conditions (bar 1: 14.17±4.79%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05. (C) LC3B conversion analysis in E. coli‐phagocytosing neutrophils (lane II), control neutrophils (lane I) and E. coli‐phagocytosing neutrophils after 3‐MA pretreatment (lane III). One representative out of six independent experiments is shown. (D) Effect of 3‐MA administration to E. coli‐phagocytosing neutrophils 15 min after the initiation of phagocytosis (lane III) as detected by LC3B conversion analysis. Lane II represents E. coli‐phagocytosing neutrophils and lane I control neutrophils. One representative out of four independent experiments is shown. (E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 6

Alterations in ATG gene expression after phagocytosis of opsonized E. coli. ATG3 (2.05±0.27), ATG4 (2.20±0.27), ATG5 (2.89±0.42), ATG6 (2.04±0.40), ATG7 (1.98±0.30) and LC3B (2.46±0.39) gene expression (fold) in E. coli‐phagocytosing neutrophils compared with control cells. Dotted line represents mRNA expression levels of control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD Wilcoxon matched‐pairs test, *p<0.05.
Regulation of the autophagic machinery in human neutrophils (2010)
Figure 7

ROS inhibition studies and their impact on neutrophil autophagic activity. (A) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E. coli stimulation (lane I: PMA, lane II: PMA+BHA, lane III: E. coli, lane IV: E. coli+BHA, lane V: control cells). One representative out of four independent experiments is shown. (B) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E. coli‐phagocytosing neutrophils (lane I: PBS, lane II: E. coli, lane III: E. coli+DPI). One representative out of four independent experiments is shown. (C) (I) Effect of NADPH‐oxidase inhibition with DPI in the formation of acidified autophagosomes in E. coli‐phagocytosing neutrophils. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli‐phagocytosing PMN (77.17±8.33%) and PMN treated with DPI before incubation with opsonized E. coli (21.67±4.37%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05 (D) (I) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan (PGN), compared with cells treated with PGN. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. PGN‐treated neutrophils (74.83±9.09%) and neutrophils pretreated with BNP before the addition of PGN (17.33±2.07%) compared with control conditions (14.67±3.20%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05, n.s.=not significant: (E) Alterations in LC3B conversion in BNP pretreated, peptidoglycan‐stimulated neutrophils (lane III), (lane I: medium‐treated cells, lane II: peptidoglycan‐treated cells). One representative out of four independent experiments is shown. (F) Effect of PI3K inhibition with 3‐MA in the percentage of PMA‐induced oxidizing cells. PMA (99.41±0.33%) and PMA+3‐MA (15.82±3.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.
USP33 Deubiquitinates and Stabilizes HIF-2alpha to Promote Hypoxia Response in Glioma Stem Cells (2022)
Figure 1

Figure 1. USP33 is a hypoxia-inducible deubiquitinase that is preferentially expressed in GSCs.

A Immunoblot analysis of USP33 and HIF2α protein levels in GSCs cultured under 5% oxygen for different times. GSCs (T387, T3359 and T4121) were cultured under 5% oxygen for 0, 12 or 24 hours before harvest. Dramatic induction of USP33 was observed at 12 hours under 5% oxygen. HIF2α protein levels were slightly upregulated at 12 hours and further elevated at 24 hours under 5% oxygen.

B Immunoblot analysis of USP33 and HIF2α protein levels in GSCs cultured in different concentrations of oxygen. GSCs were cultured under 20%, 5%, 3% or 1% oxygen for 24 hours before harvest. Induction of USP33 was observed under 5%, 3% and 1% oxygen.

C Immunofluorescent analyses of USP33 (red), the hypoxia marker CA9 (green), and the stem cell marker SOX2 (gray) in human primary GBMs. Arrows indicate the USP33+/CA9+/SOX2+ cells. Scale bar, 80 µm.

D Statistical quantification of (C) showing the ratio of USP33+ cells in SOX2+, CA9+, or SOX2+/CA9+ cells in human primary GBMs. USP33 expression was detected in a fraction of GSCs (SOX2+) or CA9+ hypoxic cells, but the majority of the hypoxic GSCs (SOX2+/CA9+ cells) were USP33+, indicating that USP33 was induced in GSCs in response to hypoxia. (n = 5 sections; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test).

E Immunofluorescent analyses of USP33 (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 hours post injection. Hypoxic cells were labeled with the FITC-conjugated mouse monoclonal anti-pimonidazole antibody. Scale bar, 80 µm.

F Statistical quantification of (E) showing the percentage of USP33+ cells in the pimonidazole+ hypoxic and the pimonidazole- normoxic cells. The majority of hypoxic cell population in GSC-derived xenografts were positively stained for USP33. (n = 5 tumors; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test).

G Immunoblot analysis of USP33, HIF2α and HIF1α protein levels in GSCs, NSTCs and NPCs in normoxic and hypoxic conditions. Hypoxia (5% O2) relative to normoxia (20% O2) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs. The mild hypoxia of 5% O2 showed negligible induction of HIF1α in all cells.