Figure S1.
Mechanosensing inhibits pDCs response without impacting cellular viability and without the involvement of the Hippo pathway. (A) Purified pDCs from HDs (n = 4) were cultured either at 0.2 or 50 kPa for 5 h and analyzed by RNA-seq. All modulated pathways were identified using QIAGEN IPA as indicated by the arrows. (B and C) Purified pDCs from HDs (n = 6; four independent experiments) were cultured in media alone or at increasing stiffness, followed by incubation with the TLR9 agonist (C274 at 0.075 μM). (B) Expression level of IFNA2 was analyzed by Q-PCR at 5 h. (C) After 15 h of culture, IL6 secretion was analyzed by ELISA. (D) Purified pDCs from HDs (n = 5; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 3, 6 and 9 h. Gene expression level of IFNA2 was analyzed by Q-PCR. (E and F) Purified pDCs from HDs (n = 3; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 6 h. Percentage of (E) cell viability or (F) of apoptotic cells (Annexin V+) was assessed by flow cytometry. (G) Purified pDCs from HDs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at either 75 or 675 nM) for 15 h. IFNα secretion was analyzed by ELISA. (H and I) RNA-seq of pDCs from HDs (Chaudhary et al., 2022) was reanalyzed for the expression of (H) integrin genes and (I) genes related to Hippo pathway and represented in CPM (count per million). (J) Purified pDCs from HDs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) alone or with super TDU-131 (100 ng/ml) (YAP inhibitor) for 15 h. IFNα secretion was analyzed by ELISA. (K) Purified pDCs from HDs were cultured at either low stiffness (0.2 kPa) or high stiffness (50 kPa) for 6 h and analyzed by RNA-seq. Differentially regulated transcriptional factors were analyzed by QIAGEN IPA and upregulated and downregulated TFs are indicated in red and blue, respectively. Individual donors are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann–Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. **P < 0.01. Refer to the image caption for details.

Mechanosensing inhibits pDCs response without impacting cellular viability and without the involvement of the Hippo pathway. (A) Purified pDCs from HDs (n = 4) were cultured either at 0.2 or 50 kPa for 5 h and analyzed by RNA-seq. All modulated pathways were identified using QIAGEN IPA as indicated by the arrows. (B and C) Purified pDCs from HDs (n = 6; four independent experiments) were cultured in media alone or at increasing stiffness, followed by incubation with the TLR9 agonist (C274 at 0.075 μM). (B) Expression level of IFNA2 was analyzed by Q-PCR at 5 h. (C) After 15 h of culture, IL6 secretion was analyzed by ELISA. (D) Purified pDCs from HDs (n = 5; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 3, 6 and 9 h. Gene expression level of IFNA2 was analyzed by Q-PCR. (E and F) Purified pDCs from HDs (n = 3; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 6 h. Percentage of (E) cell viability or (F) of apoptotic cells (Annexin V+) was assessed by flow cytometry. (G) Purified pDCs from HDs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at either 75 or 675 nM) for 15 h. IFNα secretion was analyzed by ELISA. (H and I) RNA-seq of pDCs from HDs (Chaudhary et al., 2022) was reanalyzed for the expression of (H) integrin genes and (I) genes related to Hippo pathway and represented in CPM (count per million). (J) Purified pDCs from HDs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) alone or with super TDU-131 (100 ng/ml) (YAP inhibitor) for 15 h. IFNα secretion was analyzed by ELISA. (K) Purified pDCs from HDs were cultured at either low stiffness (0.2 kPa) or high stiffness (50 kPa) for 6 h and analyzed by RNA-seq. Differentially regulated transcriptional factors were analyzed by QIAGEN IPA and upregulated and downregulated TFs are indicated in red and blue, respectively. Individual donors are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann–Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. **P < 0.01.

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