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jnk2  (Sino Biological)
93
Sino Biological jnk2
HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, <t>JNK2,</t> or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.
Jnk2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+jnk2/Human+JNK2%2FMAPK9+Gene+ORF+cDNA+clone+expression+plasmid/pmc12450640-224-8-15
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pure active human jnk2 protein  (Millipore)
90
Millipore pure active human jnk2 protein
HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, <t>JNK2,</t> or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.
Pure Active Human Jnk2 Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+jnk2/jnk+ii/pmc11664791-250-19-23
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pure active human jnk2 protein - by Bioz Stars, 2026-08
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n a recombinant human gst jnk2 signalchem  (Sino Biological)
92
Sino Biological n a recombinant human gst jnk2 signalchem
HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, <t>JNK2,</t> or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.
N A Recombinant Human Gst Jnk2 Signalchem, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+jnk2/JNK2%2C+Active/pm39454579-567-121-125
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n a recombinant human gst jnk2 signalchem - by Bioz Stars, 2026-08
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recombinant human gst jnk2  (Sino Biological)
92
Sino Biological recombinant human gst jnk2
HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, <t>JNK2,</t> or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.
Recombinant Human Gst Jnk2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+jnk2/JNK2%2C+Active/pmc11585421-918-0-4
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recombinant human gst jnk2 - by Bioz Stars, 2026-08
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human c-jun n-terminal kinase jnk2  (Atomwise Inc)
90
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jnk2 pt183 y185  (R&D Systems)
93
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t183 y185  (R&D Systems)
92
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anti human integrin α5  (R&D Systems Hematology)
91
R&D Systems Hematology anti human integrin α5
FN-rich ECM inhibits the formation of FCL and RA. (A) U2Os-AP2-GFP cells plated on PLL-, BSA-, FN-, VTN-, Col I-, Col IV-, LN111-coated, or non-coated dishes overnight. Samples were imaged with TIRF microscopy at 1-s intervals for <t>5</t> min. Representative 15-s time projections and 5-min kymographs of time-lapse videos from samples in . (B) Left: U2Os-AP2-GFP cells plated on non-coated dishes overnight were stained for FN. Representative TIRF images. Right: Graph showing the reduced brightness of AP2 in regions with higher FN signal (measured from a 1.5 µm × 1.5 µm region around each AP2 spot, n = 32988 AP2 spots, 27 images from one representative sample). (C) U2OS cells plated on non-coated (NC) or FN-coated (10 µg/ml) dishes were analyzed for clathrin exon 31 density by RT-PCR; n = 3 biological replicates. (D) U2Os-AP2-GFP were plated to non-coated dishes and stained for <t>integrin</t> αvβ5 and VTN. Representative TIRF images. (E) Analysis of integrin αvβ5 fluorescent intensity of samples from . N (images): FN = 15, VTN/Col I/LN111/non-coated = 21, Col IV = 17. Results were obtained from one representative experiment; similar results were observed in four individual experiments. F (5, 120) = 85.49, P < 0.0001. (F) U2Os-AP2-GFP cells plated on 10 µg/ml FN, 5 µg/ml FN, 1 µg/ml FN, FBS, or complete MEM medium-coated dishes were stained for FN and integrin αvβ5. Representative TIRF images. (G) FN integrated fluorescent density of dishes coated as in G, and plated or not plated with U2Os cells. N = 16-10/sample, from two independent experiments. F (9, 129) = 184.8, P < 0.0001. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 and 5 µm; insets, except in D, are 2 µm. Source data are available for this figure: .
Anti Human Integrin α5, supplied by R&D Systems Hematology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+jnk2/Human%2FMouse%2FRat+JNK2+Antibody/pmc10225744-228-29-35
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anti human integrin α5 - by Bioz Stars, 2026-08
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snaka51  (R&D Systems Hematology)
91
R&D Systems Hematology snaka51
Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 <t>(SNAKA51</t> antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .
Snaka51, supplied by R&D Systems Hematology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, JNK2, or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.

Journal: The Journal of Biological Chemistry

Article Title: Activation of the stress-activated protein kinase JNK in response to herpes simplex virus-1 infection coordinates transition of BRD4 from chromosome association to transcription elongation

doi: 10.1016/j.jbc.2025.110590

Figure Lengend Snippet: HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, JNK2, or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.

Article Snippet: The plasmids for mammalian expression of JNK1 (HG10795-UT), JNK2 (HG10745-UT), and BRD4 were purchased from SinoBiological (Beijing), pcDNA5-CMV-FLAG-BRD4 wt was purchased from Miaoling ( P29409 , Wuhan), and pcDNA5-CMV-FLAG-BRD4-T1186A/T1212A was synthesized by GenScript.

Techniques: Infection, Phospho-proteomics, Expressing, Inhibition, Control, Knock-Out, Construct, Western Blot, Virus, Transfection

Sequence conservation between the SmJNK and JNK2. (A) Sequence identity between the human JNK (JNK2) and S. mansoni JNK (SmJNK) PKD was analysed by sequence alignment in Clustal Omega ( <xref ref-type=Sievers et al., 2011 ). Conserved domains for kinase-ligand interaction were highlighted in yellow (G-rich loop, αC-helix, gatekeeper (gk) residue, hinge, and DFG motif. Activation loop is highlighted in magenta. (B) Schematic representation of the 85 amino acid residues from KLIFS database ( Van Linden et al., 2014 ) of both kinases. The differences in SmJNK compared to the human JNK are highlighted in red. (C) Ribbon and surface representation of the crystal structure of JNK2 (blue) bound to BIRB796 (orange) ( Kuglstatter et al., 2010 ). Domains highlighted in (A) are shown here in the same colors. (D) Schematic representation of kinase-ligand interactions of JNK2 and BIRB796 among the KLIFS residues. " width="100%" height="100%">

Journal: Frontiers in Parasitology

Article Title: Identification of potent schistosomicidal compounds predicted as type II-kinase inhibitors against Schistosoma mansoni c-Jun N-terminal kinase SMJNK

doi: 10.3389/fpara.2024.1394407

Figure Lengend Snippet: Sequence conservation between the SmJNK and JNK2. (A) Sequence identity between the human JNK (JNK2) and S. mansoni JNK (SmJNK) PKD was analysed by sequence alignment in Clustal Omega ( Sievers et al., 2011 ). Conserved domains for kinase-ligand interaction were highlighted in yellow (G-rich loop, αC-helix, gatekeeper (gk) residue, hinge, and DFG motif. Activation loop is highlighted in magenta. (B) Schematic representation of the 85 amino acid residues from KLIFS database ( Van Linden et al., 2014 ) of both kinases. The differences in SmJNK compared to the human JNK are highlighted in red. (C) Ribbon and surface representation of the crystal structure of JNK2 (blue) bound to BIRB796 (orange) ( Kuglstatter et al., 2010 ). Domains highlighted in (A) are shown here in the same colors. (D) Schematic representation of kinase-ligand interactions of JNK2 and BIRB796 among the KLIFS residues.

Article Snippet: The human c-Jun N-terminal kinase JNK2 (PDB ID: 3NPC) was selected by Atomwise as a template for the virtual screening.

Techniques: Sequencing, Residue, Activation Assay

Detailed analysis of the interaction of inactive SmJNK with compounds A04 and B08. (A) Ribbon and surface representation (white) of the homology models of SmJNK in active (DFG-in) and inactive (DFG-out) conformation, and crystal structure of human JNK2 (DFG-out). G-rich loop, αC-helix, and DFG motifs are colored in blue. Activation loop is colored in magenta. (B) Left: ribbon and surface representation of inactive SmJNK (grey) and docked A04 (cyan) with interacting residues highlighted in magenta. Right: Detailed representation of the docking with labeled residues and their side chains in stick representation. G-rich loop, αC-helix, and the catalytic loop are shown in white. (C) Schematic representation of KLIFS residues showing the kinase-ligand interactions highlighted in (B) . (D) Left: ribbon and surface representation of inactive SmJNK (grey) and docked B08 (cyan) with interacting residues highlighted in magenta. Right: Detailed representation of the docking with labeled residues and their side chains in stick representation. G-rich loop, αC-helix, and the catalytic loop are shown in white. (E) Schematic representation of KLIFS residues showing the kinase-ligand interactions highlighted in (D) .

Journal: Frontiers in Parasitology

Article Title: Identification of potent schistosomicidal compounds predicted as type II-kinase inhibitors against Schistosoma mansoni c-Jun N-terminal kinase SMJNK

doi: 10.3389/fpara.2024.1394407

Figure Lengend Snippet: Detailed analysis of the interaction of inactive SmJNK with compounds A04 and B08. (A) Ribbon and surface representation (white) of the homology models of SmJNK in active (DFG-in) and inactive (DFG-out) conformation, and crystal structure of human JNK2 (DFG-out). G-rich loop, αC-helix, and DFG motifs are colored in blue. Activation loop is colored in magenta. (B) Left: ribbon and surface representation of inactive SmJNK (grey) and docked A04 (cyan) with interacting residues highlighted in magenta. Right: Detailed representation of the docking with labeled residues and their side chains in stick representation. G-rich loop, αC-helix, and the catalytic loop are shown in white. (C) Schematic representation of KLIFS residues showing the kinase-ligand interactions highlighted in (B) . (D) Left: ribbon and surface representation of inactive SmJNK (grey) and docked B08 (cyan) with interacting residues highlighted in magenta. Right: Detailed representation of the docking with labeled residues and their side chains in stick representation. G-rich loop, αC-helix, and the catalytic loop are shown in white. (E) Schematic representation of KLIFS residues showing the kinase-ligand interactions highlighted in (D) .

Article Snippet: The human c-Jun N-terminal kinase JNK2 (PDB ID: 3NPC) was selected by Atomwise as a template for the virtual screening.

Techniques: Activation Assay, Labeling

FN-rich ECM inhibits the formation of FCL and RA. (A) U2Os-AP2-GFP cells plated on PLL-, BSA-, FN-, VTN-, Col I-, Col IV-, LN111-coated, or non-coated dishes overnight. Samples were imaged with TIRF microscopy at 1-s intervals for 5 min. Representative 15-s time projections and 5-min kymographs of time-lapse videos from samples in . (B) Left: U2Os-AP2-GFP cells plated on non-coated dishes overnight were stained for FN. Representative TIRF images. Right: Graph showing the reduced brightness of AP2 in regions with higher FN signal (measured from a 1.5 µm × 1.5 µm region around each AP2 spot, n = 32988 AP2 spots, 27 images from one representative sample). (C) U2OS cells plated on non-coated (NC) or FN-coated (10 µg/ml) dishes were analyzed for clathrin exon 31 density by RT-PCR; n = 3 biological replicates. (D) U2Os-AP2-GFP were plated to non-coated dishes and stained for integrin αvβ5 and VTN. Representative TIRF images. (E) Analysis of integrin αvβ5 fluorescent intensity of samples from . N (images): FN = 15, VTN/Col I/LN111/non-coated = 21, Col IV = 17. Results were obtained from one representative experiment; similar results were observed in four individual experiments. F (5, 120) = 85.49, P < 0.0001. (F) U2Os-AP2-GFP cells plated on 10 µg/ml FN, 5 µg/ml FN, 1 µg/ml FN, FBS, or complete MEM medium-coated dishes were stained for FN and integrin αvβ5. Representative TIRF images. (G) FN integrated fluorescent density of dishes coated as in G, and plated or not plated with U2Os cells. N = 16-10/sample, from two independent experiments. F (9, 129) = 184.8, P < 0.0001. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 and 5 µm; insets, except in D, are 2 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: FN-rich ECM inhibits the formation of FCL and RA. (A) U2Os-AP2-GFP cells plated on PLL-, BSA-, FN-, VTN-, Col I-, Col IV-, LN111-coated, or non-coated dishes overnight. Samples were imaged with TIRF microscopy at 1-s intervals for 5 min. Representative 15-s time projections and 5-min kymographs of time-lapse videos from samples in . (B) Left: U2Os-AP2-GFP cells plated on non-coated dishes overnight were stained for FN. Representative TIRF images. Right: Graph showing the reduced brightness of AP2 in regions with higher FN signal (measured from a 1.5 µm × 1.5 µm region around each AP2 spot, n = 32988 AP2 spots, 27 images from one representative sample). (C) U2OS cells plated on non-coated (NC) or FN-coated (10 µg/ml) dishes were analyzed for clathrin exon 31 density by RT-PCR; n = 3 biological replicates. (D) U2Os-AP2-GFP were plated to non-coated dishes and stained for integrin αvβ5 and VTN. Representative TIRF images. (E) Analysis of integrin αvβ5 fluorescent intensity of samples from . N (images): FN = 15, VTN/Col I/LN111/non-coated = 21, Col IV = 17. Results were obtained from one representative experiment; similar results were observed in four individual experiments. F (5, 120) = 85.49, P < 0.0001. (F) U2Os-AP2-GFP cells plated on 10 µg/ml FN, 5 µg/ml FN, 1 µg/ml FN, FBS, or complete MEM medium-coated dishes were stained for FN and integrin αvβ5. Representative TIRF images. (G) FN integrated fluorescent density of dishes coated as in G, and plated or not plated with U2Os cells. N = 16-10/sample, from two independent experiments. F (9, 129) = 184.8, P < 0.0001. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 and 5 µm; insets, except in D, are 2 µm. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction

FN inhibits RA formation in a local manner. (A) U2Os-AP2-GFP cells plated on FN, VTN, Col I, Col IV, LN111 (all 10 µg/ml) -coated or non-coated dishes, stained for p-Pax and integrin αvβ. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N = 21 from two independent experiments. F (5, 120) = 48.05, P < 0.0001. (C) U2Os-AP2-GFP cells were plated on FN/glass patterned imaging dishes overnight, stained for integrin αvβ5 and p-Pax, and imaged using TIRF microscopy; representative TIRF images. (D) Quantification of integrin αvβ5 fluorescent intensity on FN- and glass-side of the pattern; n = 10 from one representative experiment. Similar results were observed in five similar experiments. Two-tailed Student’s t test, P < 0.01. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: FN inhibits RA formation in a local manner. (A) U2Os-AP2-GFP cells plated on FN, VTN, Col I, Col IV, LN111 (all 10 µg/ml) -coated or non-coated dishes, stained for p-Pax and integrin αvβ. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N = 21 from two independent experiments. F (5, 120) = 48.05, P < 0.0001. (C) U2Os-AP2-GFP cells were plated on FN/glass patterned imaging dishes overnight, stained for integrin αvβ5 and p-Pax, and imaged using TIRF microscopy; representative TIRF images. (D) Quantification of integrin αvβ5 fluorescent intensity on FN- and glass-side of the pattern; n = 10 from one representative experiment. Similar results were observed in five similar experiments. Two-tailed Student’s t test, P < 0.01. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Staining, Imaging, Microscopy, Two Tailed Test

FCLs and RAs presence correlates with FN production in multiple cells lines. (A–E) The following knock-in cell lines were used in this figure: U2Os-AP2-GFP, HeLa-AP2-GFP, hMEC-AP2-GFP, U2Os-AP2-halo, HeLa-AP2-halo, MCF7-AP2-halo, HDF-AP2-halo, and Caco2-AP2-halo. (A) Cell lines indicated were plated to non-coated (nc) dishes, allowed to settle overnight, and stained for integrin αvβ5 and FN. Representative TIRF images. (B) Analysis of FN fluorescent intensity from samples in A. N (images): U2Os = 21, HeLa = 20, MCF7 = 23, HDF = 33, Caco2 = 16, hMEC = 23, from two independent experiments. F (5, 130) = 56.16, P < 0.0001. (C) Cells were plated on FN-coated or nc dishes, allowed to settle overnight, and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C. Cell lines with an AP2 halo tag, n (images): U2Os (nc/FN) = 20, HeLa (nc) = 33, HeLa (FN) = 29, MCF7 (nc) = 29, MCF7 (FN) = 32, HDF (nc/FN) 32, Caco2 (nc/FN) = 32. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (9, 273) = 64.96, P < 0.0001. (E) Analysis of RA coverage from samples in C. Cell lines with an AP2 GFP tag, n (images): U2Os (nc) = 31, U2Os (FN) = 18, HeLa (nc) = 35, HeLa (FN) = 38, and hMEC (nc/FN) = 38. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (5, 189) = 89.97, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: FCLs and RAs presence correlates with FN production in multiple cells lines. (A–E) The following knock-in cell lines were used in this figure: U2Os-AP2-GFP, HeLa-AP2-GFP, hMEC-AP2-GFP, U2Os-AP2-halo, HeLa-AP2-halo, MCF7-AP2-halo, HDF-AP2-halo, and Caco2-AP2-halo. (A) Cell lines indicated were plated to non-coated (nc) dishes, allowed to settle overnight, and stained for integrin αvβ5 and FN. Representative TIRF images. (B) Analysis of FN fluorescent intensity from samples in A. N (images): U2Os = 21, HeLa = 20, MCF7 = 23, HDF = 33, Caco2 = 16, hMEC = 23, from two independent experiments. F (5, 130) = 56.16, P < 0.0001. (C) Cells were plated on FN-coated or nc dishes, allowed to settle overnight, and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C. Cell lines with an AP2 halo tag, n (images): U2Os (nc/FN) = 20, HeLa (nc) = 33, HeLa (FN) = 29, MCF7 (nc) = 29, MCF7 (FN) = 32, HDF (nc/FN) 32, Caco2 (nc/FN) = 32. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (9, 273) = 64.96, P < 0.0001. (E) Analysis of RA coverage from samples in C. Cell lines with an AP2 GFP tag, n (images): U2Os (nc) = 31, U2Os (FN) = 18, HeLa (nc) = 35, HeLa (FN) = 38, and hMEC (nc/FN) = 38. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (5, 189) = 89.97, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Knock-In, Staining

Integrin αvβ5 is necessary in the formation of FCL. (A) U2Os-AP2-GFP/halo, HeLa-AP2-GFP/halo, and MCF7-AP2-halo were plated on FN-coated or non-coated dishes, allowed to settle overnight, stained for integrin αvβ5 and p-Pax, imaged using TIRF, and analyzed for FA coverage. N (images): U2Os glass/FN = 37, HeLa glass = 32, Hela FN = 29, MCF7 glass = 28, and MCF7 FN = 32. (B) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control were imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (C) Analysis of FCL proportions from time-lapse videos in A. N (videos): control = 8, shITGB5 #1 = 11, shITGB5 #2 = 6, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 22) = 44.46, P < 0.0001. (D) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control, were stained for integrin αvβ5 and p-Pax. Representative TIRF images. (E) Analysis of integrin β5 silencing efficiency, n = 10, from one representative experiment. Similar results were observed from three independent experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 27) = 63.29, P < 0.0001. (F) U2Os-AP2-GFP cells plated on non-coated dishes for 20 h were treated with the integrin αvβ5 inhibitor Cilengitide (10 µM) for 15 or 45 min and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (G) Analysis of AP2 signal colocalizing with p-Pax or integrin αvβ5 over time from samples in E. N (images): control = 14, Cil 15 min = 13, Cil 45 min = 10, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA and Tukey’s multiple comparison F (2, 99) = 63.38, P < 0.0001. Data are the mean ± SD, ns. non-significant P -value; *** P -value < 0.001. Scale bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Integrin αvβ5 is necessary in the formation of FCL. (A) U2Os-AP2-GFP/halo, HeLa-AP2-GFP/halo, and MCF7-AP2-halo were plated on FN-coated or non-coated dishes, allowed to settle overnight, stained for integrin αvβ5 and p-Pax, imaged using TIRF, and analyzed for FA coverage. N (images): U2Os glass/FN = 37, HeLa glass = 32, Hela FN = 29, MCF7 glass = 28, and MCF7 FN = 32. (B) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control were imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (C) Analysis of FCL proportions from time-lapse videos in A. N (videos): control = 8, shITGB5 #1 = 11, shITGB5 #2 = 6, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 22) = 44.46, P < 0.0001. (D) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control, were stained for integrin αvβ5 and p-Pax. Representative TIRF images. (E) Analysis of integrin β5 silencing efficiency, n = 10, from one representative experiment. Similar results were observed from three independent experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 27) = 63.29, P < 0.0001. (F) U2Os-AP2-GFP cells plated on non-coated dishes for 20 h were treated with the integrin αvβ5 inhibitor Cilengitide (10 µM) for 15 or 45 min and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (G) Analysis of AP2 signal colocalizing with p-Pax or integrin αvβ5 over time from samples in E. N (images): control = 14, Cil 15 min = 13, Cil 45 min = 10, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA and Tukey’s multiple comparison F (2, 99) = 63.38, P < 0.0001. Data are the mean ± SD, ns. non-significant P -value; *** P -value < 0.001. Scale bars, 10 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Staining, Control, Microscopy, Comparison

Inhibition of CME prevents RA formation. (A) U2Os-AP2-GFP cells silenced for AP2A1, AP2S1, or control (scrambled shRNA) were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N (images): control = 12, shAP2A1#1 = 8, shAPA1#2 = 11, shAPS1#1 = 10, shAPS1#2 = 19, shAPS1#3 = 10. Data were obtained from two individual experiments and similar results were observed in five independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (5, 64) = 43.11, P < 0.001. (C) U2Os-AP2-GFP cells overexpressing Ap180 ct were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C; n = 17 from one representative experiment and similar results were observed in four individual experiments. Two-tailed Student’s t test, P < 0.0001. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Inhibition of CME prevents RA formation. (A) U2Os-AP2-GFP cells silenced for AP2A1, AP2S1, or control (scrambled shRNA) were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N (images): control = 12, shAP2A1#1 = 8, shAPA1#2 = 11, shAPS1#1 = 10, shAPS1#2 = 19, shAPS1#3 = 10. Data were obtained from two individual experiments and similar results were observed in five independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (5, 64) = 43.11, P < 0.001. (C) U2Os-AP2-GFP cells overexpressing Ap180 ct were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C; n = 17 from one representative experiment and similar results were observed in four individual experiments. Two-tailed Student’s t test, P < 0.0001. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Inhibition, Control, shRNA, Staining, Comparison, Two Tailed Test

RAs are formed at FCLs. (A) U2Os-AP2-ITGB5-mScarlet cells plated on glass were treated with Cilengitide (10 µM) for 15 min, washed twice with fresh medium, and imaged live using TIRF microscopy at two frames per minute. Representative frames showing the growth of an RA from an FCL. (B) Analysis of AP2 and integrin αvβ5 (ITGB5) intensity over time. Left: FCL and αvβ5 clustering during RA assembly ( n = 16 events). In this graph, time zero is defined as the arrival of the AP2 signal Right: ITGB5 clusters not stabilized by FCLs disassemble and are removed from the membrane by CME-mediated disassembly ( n = 24 events). In this graph, time zero is defined as the disappearance of the ITGB5 signal. Events have been collected from four 1-h long time-lapse acquisitions. Mean ± SEM. (C) A schematic illustration of the results is shown in this figure. Scale bar, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: RAs are formed at FCLs. (A) U2Os-AP2-ITGB5-mScarlet cells plated on glass were treated with Cilengitide (10 µM) for 15 min, washed twice with fresh medium, and imaged live using TIRF microscopy at two frames per minute. Representative frames showing the growth of an RA from an FCL. (B) Analysis of AP2 and integrin αvβ5 (ITGB5) intensity over time. Left: FCL and αvβ5 clustering during RA assembly ( n = 16 events). In this graph, time zero is defined as the arrival of the AP2 signal Right: ITGB5 clusters not stabilized by FCLs disassemble and are removed from the membrane by CME-mediated disassembly ( n = 24 events). In this graph, time zero is defined as the disappearance of the ITGB5 signal. Events have been collected from four 1-h long time-lapse acquisitions. Mean ± SEM. (C) A schematic illustration of the results is shown in this figure. Scale bar, 5 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Microscopy, Membrane

Integrin β1 blocking stimulates FCL and RA formation. (A) U2Os-AP2-GFP cells plated on FN were treated with integrin β1 blocking antibody mab13 (0.3 µg/ml) for 15 and 45 min (or vehicle for 45 min = control) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of AP2 signal colocalizing with no markers (non), p-Pax, or integrin αvβ5 over time. N (images): control = 18, mab13 15 min = 13, mab13 45 min = 15, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 119) = 46.68, P < 0.0001. (C) Analysis of fraction of integrin αvβ5 colocalizing with AP2 over time from samples in A. N (images): control = 15, mab13 15 min = 11, mab13 45 min = 12, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 35) = 45.81, P < 0.0001. (D) U2Os-AP2-GFP cells plated on FN were treated with mab13 (0.3 µg/ml) and imaged using TIRF microscopy. 5-min time-lapses with 1-s intervals starting at 0 min (no mab13) and every 5 min after mab13 addition, until 35 min, were acquired. N (videos): 0 min = 11, 5 min = 11, 10 min = 9, 15 min = 8, 20 min = 8, 25 min = 6, 30 min = 7, 35 min = 6. Videos were acquired from two independent experiments. Similar results were observed in five individual experiments. One-way ANOVA with Tukey’s multiple comparison. F (7, 29) = 8.893, P < 0.0001. (E) A schematic illustration of results shown in this figure. Data are the mean ± SD, ns. non-significant P value; * P value<0.05, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Integrin β1 blocking stimulates FCL and RA formation. (A) U2Os-AP2-GFP cells plated on FN were treated with integrin β1 blocking antibody mab13 (0.3 µg/ml) for 15 and 45 min (or vehicle for 45 min = control) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of AP2 signal colocalizing with no markers (non), p-Pax, or integrin αvβ5 over time. N (images): control = 18, mab13 15 min = 13, mab13 45 min = 15, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 119) = 46.68, P < 0.0001. (C) Analysis of fraction of integrin αvβ5 colocalizing with AP2 over time from samples in A. N (images): control = 15, mab13 15 min = 11, mab13 45 min = 12, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 35) = 45.81, P < 0.0001. (D) U2Os-AP2-GFP cells plated on FN were treated with mab13 (0.3 µg/ml) and imaged using TIRF microscopy. 5-min time-lapses with 1-s intervals starting at 0 min (no mab13) and every 5 min after mab13 addition, until 35 min, were acquired. N (videos): 0 min = 11, 5 min = 11, 10 min = 9, 15 min = 8, 20 min = 8, 25 min = 6, 30 min = 7, 35 min = 6. Videos were acquired from two independent experiments. Similar results were observed in five individual experiments. One-way ANOVA with Tukey’s multiple comparison. F (7, 29) = 8.893, P < 0.0001. (E) A schematic illustration of results shown in this figure. Data are the mean ± SD, ns. non-significant P value; * P value<0.05, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Blocking Assay, Control, Staining, Comparison, Microscopy

Depletion of integrin β1 or Integrin α5 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax and imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (B) Analysis of FCL proportions from samples in A. N (videos): shScr = 12, shITGB1 #1 = 11, shITGB1 #2 = 10, from three individual experiments. Tukey’s multiple comparison, F (2, 30) = 27.81, P < 0.001. (C) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB #1, #2), or integrin α5 with two different shRNAs (shITGA5 #1, #2), or control shRNA, were plated on FN-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from integrin β1 silenced samples in C. N (images): control shRNA = 30, shITGB1 #1 = 27, shITGB1 #2 = 18, from two independent experiments; similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 72) = 276.2, P < 0.0001. (E) Analysis of RA coverage from integrin α5 silenced samples in E. N (images): control shRNA = 33, shITGA5 #1 = 40, shITGA5 #2 = 37, from three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 107) = 44.46, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P-value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Depletion of integrin β1 or Integrin α5 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax and imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (B) Analysis of FCL proportions from samples in A. N (videos): shScr = 12, shITGB1 #1 = 11, shITGB1 #2 = 10, from three individual experiments. Tukey’s multiple comparison, F (2, 30) = 27.81, P < 0.001. (C) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB #1, #2), or integrin α5 with two different shRNAs (shITGA5 #1, #2), or control shRNA, were plated on FN-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from integrin β1 silenced samples in C. N (images): control shRNA = 30, shITGB1 #1 = 27, shITGB1 #2 = 18, from two independent experiments; similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 72) = 276.2, P < 0.0001. (E) Analysis of RA coverage from integrin α5 silenced samples in E. N (images): control shRNA = 33, shITGA5 #1 = 40, shITGA5 #2 = 37, from three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 107) = 44.46, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P-value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Control, shRNA, Staining, Microscopy, Comparison

Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Control, shRNA, Staining, Comparison, Western Blot, Molecular Weight

Active integrin α5β1 at fibrillar adhesions inhibit FCL and RA formation. (A) U2Os-AP2-halo cells were plated to FN-coated or non-coated dishes and stained for Tensin1 and active integrin β1 12G10. Representative TIRF images. (B) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (C) Analysis of RA coverage from samples in B. N (images): shScr control = 35, shTNS1 #1/#2 = 44, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 120) = 56.26, P < 0.0001. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (E) Analysis of 12G10 fluorescent intensity from samples in D. N (images): shScr = 20, shTNS1 #1 = 15, shTNS1 #2 = 11, from one representative image. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 43) = 87.81, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Active integrin α5β1 at fibrillar adhesions inhibit FCL and RA formation. (A) U2Os-AP2-halo cells were plated to FN-coated or non-coated dishes and stained for Tensin1 and active integrin β1 12G10. Representative TIRF images. (B) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (C) Analysis of RA coverage from samples in B. N (images): shScr control = 35, shTNS1 #1/#2 = 44, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 120) = 56.26, P < 0.0001. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (E) Analysis of 12G10 fluorescent intensity from samples in D. N (images): shScr = 20, shTNS1 #1 = 15, shTNS1 #2 = 11, from one representative image. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 43) = 87.81, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Staining, Control, shRNA, Comparison

Knockdown of focal or fibrillar adhesion components or lateral confinement promote FCL and RA formation. (A) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for Tensin1. Representative TIRF images. (B) Analysis of Tensin1 fluorescent intensity from samples in A. N (images): shScr control = 22, shTNS1 #1 = 29, shTNS1 #2 = 31, from one representative experiment. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 79) = 38.09, P < 0.0001. (C) Representative Western blots of Tensin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were blotted for Tensin1 and α-tubulin. The arrow marks the correct Tensin1 band. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for Talin1. Representative TIRF images. (E) Analysis of Talin1 fluorescent intensity from samples in D. N (images): shScr = 32, shTLN1 = 31, from two individual experiments. Two-tailed Student’s t test, P < 0.0001. (F) Representative Western blots of Talin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 shRNA or control shRNA were blotted for Talin1 and GAPDH. The position of molecular weight markers (in kDa) are shown on the left. (G) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (H) Analysis of RA coverage from samples in G. N (images): n = 23. Two-tailed Student’s t-test, P < 0.0001. (I) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for integrin β1 12G10. Representative TIRF images. (J) Analysis of 12G10 fluorescent intensity with samples from I. N (images): shScr = 20, shTLN1 = 15, from one representative experiment. Similar results were observed in two individual experiments. Two-tailed Student’s t test, P < 0.0004. (K) U2Os-AP2-GFP cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (L) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. Data are the mean ± SD, ** P < 0.01, *** P < 0.001. Scale bars, 10 µm; insets, 5 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Knockdown of focal or fibrillar adhesion components or lateral confinement promote FCL and RA formation. (A) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for Tensin1. Representative TIRF images. (B) Analysis of Tensin1 fluorescent intensity from samples in A. N (images): shScr control = 22, shTNS1 #1 = 29, shTNS1 #2 = 31, from one representative experiment. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 79) = 38.09, P < 0.0001. (C) Representative Western blots of Tensin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were blotted for Tensin1 and α-tubulin. The arrow marks the correct Tensin1 band. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for Talin1. Representative TIRF images. (E) Analysis of Talin1 fluorescent intensity from samples in D. N (images): shScr = 32, shTLN1 = 31, from two individual experiments. Two-tailed Student’s t test, P < 0.0001. (F) Representative Western blots of Talin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 shRNA or control shRNA were blotted for Talin1 and GAPDH. The position of molecular weight markers (in kDa) are shown on the left. (G) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (H) Analysis of RA coverage from samples in G. N (images): n = 23. Two-tailed Student’s t-test, P < 0.0001. (I) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for integrin β1 12G10. Representative TIRF images. (J) Analysis of 12G10 fluorescent intensity with samples from I. N (images): shScr = 20, shTLN1 = 15, from one representative experiment. Similar results were observed in two individual experiments. Two-tailed Student’s t test, P < 0.0004. (K) U2Os-AP2-GFP cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (L) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. Data are the mean ± SD, ** P < 0.01, *** P < 0.001. Scale bars, 10 µm; insets, 5 µm. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Knockdown, Control, shRNA, Staining, Comparison, Western Blot, Molecular Weight, Two Tailed Test

RA disassembly is coupled to cell migration. (A) U2Os-AP2-GFP cells were grown on FN-precoated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. See for the same staining of H-shaped patterns. (B) Analysis of RA coverage from samples in A. N (images): Arrow non-coated = 28, Arrow FN = 27, H non-coated = 25, H FN = 20 from one representative image. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (C) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. See for the same staining with H-shaped patterns. (D ) Top: U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 80 min or 4 h in fresh complete medium, and stained for p-Pax. Representative TIRF images (stitched tile of five side-by-side fields of view). The wound is exactly at the right edge of the images. Bottom: Analysis of normalized RA coverage from tiles in D. RA coverage was calculated in 11-µm-wide sliding windows from the wound edge. N (tiles): 0 min = 48, 80 min = 36, 4 h = 15. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (E) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 30 min or 80 min in fresh complete medium, and stained for FN. Representative TIRF images from the migrating front (indicated as yellow lines). (F) Analysis of FN fluorescent intensity from samples in E. N (images): 0 min = 10, 30 min = 25, 80 min = 22. One-way ANOVA with Tukey’s multiple comparison, F (2, 56) = 13.77, P < 0.0001. (G) A schematic illustration of results shown in this figure. Data are the mean ± SD, ** P value < 0.01, *** P value < 0.001. Scale bars, 10 µm, except in D (50 µm) and inset (10 µm).

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: RA disassembly is coupled to cell migration. (A) U2Os-AP2-GFP cells were grown on FN-precoated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. See for the same staining of H-shaped patterns. (B) Analysis of RA coverage from samples in A. N (images): Arrow non-coated = 28, Arrow FN = 27, H non-coated = 25, H FN = 20 from one representative image. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (C) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. See for the same staining with H-shaped patterns. (D ) Top: U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 80 min or 4 h in fresh complete medium, and stained for p-Pax. Representative TIRF images (stitched tile of five side-by-side fields of view). The wound is exactly at the right edge of the images. Bottom: Analysis of normalized RA coverage from tiles in D. RA coverage was calculated in 11-µm-wide sliding windows from the wound edge. N (tiles): 0 min = 48, 80 min = 36, 4 h = 15. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (E) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 30 min or 80 min in fresh complete medium, and stained for FN. Representative TIRF images from the migrating front (indicated as yellow lines). (F) Analysis of FN fluorescent intensity from samples in E. N (images): 0 min = 10, 30 min = 25, 80 min = 22. One-way ANOVA with Tukey’s multiple comparison, F (2, 56) = 13.77, P < 0.0001. (G) A schematic illustration of results shown in this figure. Data are the mean ± SD, ** P value < 0.01, *** P value < 0.001. Scale bars, 10 µm, except in D (50 µm) and inset (10 µm).

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Migration, Staining, Comparison

Illustration of the RA coverage calculation method. To calculate RA coverage, images of the integrin (αvβ5) and paxillin (p-Pax) channels are segmented using the Robust Automatic Threshold Selection method in ImageJ. This method generates binary masks for each channel. The paxillin channel is then subtracted from the integrin channel and results in a mask containing only the RAs. RA coverage is calculated by dividing the area covered by RAs in pixels (integrated density/255) by the area covered by the cell (or cells) in an image.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Illustration of the RA coverage calculation method. To calculate RA coverage, images of the integrin (αvβ5) and paxillin (p-Pax) channels are segmented using the Robust Automatic Threshold Selection method in ImageJ. This method generates binary masks for each channel. The paxillin channel is then subtracted from the integrin channel and results in a mask containing only the RAs. RA coverage is calculated by dividing the area covered by RAs in pixels (integrated density/255) by the area covered by the cell (or cells) in an image.

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Selection

Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Control, shRNA, Staining, Comparison, Western Blot, Molecular Weight