Journal: The Journal of Biological Chemistry

Article Title: Structure-based virtual screening identifies a small-molecule inhibitor of the profilin 1–actin interaction

doi: 10.1074/jbc.M117.809137

Figure Lengend Snippet: Effects of C1 and C2 on actin polymerization with or without Pfn1 in vitro. A, Coomassie staining of an SDS-PAGE showing the purity of actin and GST–Pfn1 used in the pyrene–actin assay. B and C, pyrene–actin polymerization assay curves for the indicated experimental conditions (B, C1; C, C2) recorded for 30 min after addition of the polymerization buffer. Each time point represents the mean ± S.D. values of the fluorescence intensity of polymerized pyrene–actin relative to the maximum fluorescence intensity for the actin alone condition (data are summarized from n = 3 experiments). The insets show the chemical structures of the two compounds. The numbers in parentheses indicate relative concentrations of actin, GST–Pfn1, and the compounds. The actual concentrations of actin and Pfn1 were 10 μm and 40 μm, respectively. C1 or C2 was added either at a 50 μm (Pfn1:compound = 1:1.25) or 100 μm (Pfn1:compound = 1:2.5) concentration.

Article Snippet: Actin polymerization with or without GST–Pfn1 in the presence of DMSO or compound was performed using a fluorescence actin polymerization kit (Cytoskeleton, BK003) according to the protocol of the manufacturer, downscaled to a 96-well format with a 10:1 ratio (9 μ m unlabeled and 1 μ m pyrene actin) between unlabeled and pyrene actin.

Techniques: In Vitro, Staining, SDS Page, Pyrene Actin Assay, Polymerization Assay, Fluorescence, Concentration Assay

Journal: Endocrinology

Article Title: CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization

doi: 10.1210/en.2018-01097

Figure Lengend Snippet: Expression and distribution of MT −TIPs in Sertoli cells. (A) Using primer pairs specific to CAMSAP1, CAMSAP2, and CAMSAP3 and S16 (which served as an internal PCR control) (Table 2) for RT-PCR, these −TIPs were found to be expressed in Sertoli cells (SCs) and germ cells (GCs) in adult rat testes (T), in which brain (B) served as a positive control. The authenticity of the PCR products was confirmed by direct nucleotide sequencing. Results represent findings of n = 3 experiments that yielded similar results. Uncropped DNA gels are shown in an online repository (30). (B) A study by IB analysis using an anti-CAMSAP1 or anti-CAMSAP2 antibody (see “Materials and Methods”) illustrated only a prominent protein band with an electrophoretic mobility corresponding to CAMSAP1 or CAMSAP2. Lysates of Sertoli cells (SCs), germ cells (GCs), or testes (T) at 40 µg of protein per lane were used. (C) Schematic drawing summarizes the results of IF microscopy to visualize CAMSAP2 vs CAMSAP1 in rat or human Sertoli cells at the minus-end of MTs as noted in (D). (D) CAMSAP2 (green fluorescence) and EB1 (red fluorescence), which are a −TIP and +TIP, respectively, were costained with α-tubulin (red fluorescence, which, together with β-tubulin, forms the α-/β-dimers, which are building blocks of MTs) in rat (top three panels) or human (bottom panel) Sertoli cells. CAMSAP2 (a −TIP) and EB1 (a +TIP) appeared as long and short rodlike ultrastructures, respectively, along the MTs and were not colocalized, consistent with their functional roles, because they were preferentially localized to the corresponding minus-ends and plus-ends of the MTs (see yellow boxed area in the first, third, and fifth columns, which is enlarged in the inset in the last column of the first row). Results are representative of n = 3 experiments using different batches of Sertoli cells. Scale bars, 40 µm (10 µm in yellow inset), which apply to corresponding images in the same panel for rat or human Sertoli cells.

Article Snippet: RT-PCR data reported herein are representative findings from an experiment of n = 3 experiments. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene Primer Sequence Position Length, bp Temp., °C Cycle No. GenBank Accession No. Camsap1 Sense: 5′-CTATCGGCGAGAACACCTCTC-3′ 1065–1085 166 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001168549.1","term_id":"270483893","term_text":"NM_001168549.1"}} NM_001168549.1 Antisense: 5′-CATCGATGGCACCTCCTTCA-3′ 1211–1230 Camsap2 Sense: 5′-CGCCTTGAAAGATGGGGGAT -3′ 665–684 172 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001134503.1","term_id":"197386167","term_text":"NM_001134503.1"}} NM_001134503.1 Antisense: 5′-CGAAGTTCCTCTGGCACAGT-3′ 817–836 Camsap3 Sense: 5′-GGTTACTCCTGGGTCCTGTCT-3′ 3970–3990 139 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001144840.1","term_id":"221325615","term_text":"NM_001144840.1"}} NM_001144840.1 Antisense: 5′-GGACCATGTCTCAGCTTCTCTTT-3′ 4086-4108 S16 Sense: 5′-TCCGCTGCAGTCCGTTCAAGTCTT-3′ 15–38 385 {"type":"entrez-nucleotide","attrs":{"text":"XM_341815","term_id":"293355792","term_text":"XM_341815"}} XM_341815 Antisense: 5′-GCCAAACTTCTTGGTTTCGCAGCG-3′ 376–399 Open in a separate window Primers Used for RT-PCR Experiments MT spin-down assay to assess the relative levels of polymerized MTs The MT spin-down assay was performed as earlier described ( 22 ) to estimate the relative levels of polymerized MTs vs free ( i.e. , nonpolymerized) tubulins in Sertoli cell lysates in treatment vs control groups using kits (catalog no. BK038) from Cytoskeleton (Denver, CO) and protocols from the manufacturer.

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Positive Control, Sequencing, Microscopy, Fluorescence, Functional Assay

Journal: Endocrinology

Article Title: CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization

doi: 10.1210/en.2018-01097

Figure Lengend Snippet: Study to assess the effects of CAMSAP2 KD on the Sertoli cell TJ permeability function in vitro. (A) Regimen used to obtain samples for different analyses including RT-PCR, IB/IF analysis, or TJ permeability function by monitoring TER across the cell epithelium of n = 3 experiments, which yielded similar results. (B) RT-PCR using primer pairs specific to corresponding target genes vs S16, which served as a PCR loading control (Table 2), to assess specific KD of Camsap2 by using Camsap2-specific siRNA duplexes, which had no effects on the steady-state mRNA of either Camsap1 or Camsap3. Uncropped DNA gels are shown in an online repository (30). (C) TER measurement to assess changes in TJ permeability barrier across the Sertoli cell epithelium, illustrating that KD promoted the TJ barrier function. *P < 0.05, by Student t test, when compared with the corresponding controls of a representative experiment with n = 4 bicameral units from n = 3 experiments using different batches of Sertoli cells, which yielded similar results. (D) Results of a representative IB experiment wherein CAMSAP2 was silenced by ∼80% (see composite data of n = 3 experiments in the bar graph below, lower left panel). The steady-state protein levels of other BTB-associated proteins, including CAMSAP1, were not affected, illustrating that there were no apparent off-target effects. Data in the bar graph represent means ± SD of n = 3 experiments. The efficacy of KD was also confirmed by staining Sertoli cells for CAMSAP2 (green fluorescence), wherein cells had been transfected with CAMSAP2 siRNA duplexes vs nontargeting negative control siRNA duplexes (see lower middle panel) and is noted in the bar graph in the lower right panel. Data in the bar graph represent means ± SD of n = 3 experiments. Uncropped immunoblots are shown in an online repository (30). *P < 0.05, by Student t test. (E) When CAMSAP2 was silenced considerably as noted in (D), relative distribution of TJ (e.g., CAR, ZO-1) and basal ES (e.g., N-cadherin, β-catenin) proteins were not perturbed. Successful transfection was confirmed by siGLO (red fluorescence) detected near the cell nuclei (DAPI). Scale bars, 40 µm, which applies to other images. Results shown are representative data of an experiment from n = 3 experiments. Ctrl, control.

Article Snippet: RT-PCR data reported herein are representative findings from an experiment of n = 3 experiments. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene Primer Sequence Position Length, bp Temp., °C Cycle No. GenBank Accession No. Camsap1 Sense: 5′-CTATCGGCGAGAACACCTCTC-3′ 1065–1085 166 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001168549.1","term_id":"270483893","term_text":"NM_001168549.1"}} NM_001168549.1 Antisense: 5′-CATCGATGGCACCTCCTTCA-3′ 1211–1230 Camsap2 Sense: 5′-CGCCTTGAAAGATGGGGGAT -3′ 665–684 172 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001134503.1","term_id":"197386167","term_text":"NM_001134503.1"}} NM_001134503.1 Antisense: 5′-CGAAGTTCCTCTGGCACAGT-3′ 817–836 Camsap3 Sense: 5′-GGTTACTCCTGGGTCCTGTCT-3′ 3970–3990 139 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001144840.1","term_id":"221325615","term_text":"NM_001144840.1"}} NM_001144840.1 Antisense: 5′-GGACCATGTCTCAGCTTCTCTTT-3′ 4086-4108 S16 Sense: 5′-TCCGCTGCAGTCCGTTCAAGTCTT-3′ 15–38 385 {"type":"entrez-nucleotide","attrs":{"text":"XM_341815","term_id":"293355792","term_text":"XM_341815"}} XM_341815 Antisense: 5′-GCCAAACTTCTTGGTTTCGCAGCG-3′ 376–399 Open in a separate window Primers Used for RT-PCR Experiments MT spin-down assay to assess the relative levels of polymerized MTs The MT spin-down assay was performed as earlier described ( 22 ) to estimate the relative levels of polymerized MTs vs free ( i.e. , nonpolymerized) tubulins in Sertoli cell lysates in treatment vs control groups using kits (catalog no. BK038) from Cytoskeleton (Denver, CO) and protocols from the manufacturer.

Techniques: Permeability, In Vitro, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Fluorescence, Transfection, Negative Control, Western Blot

Journal: Endocrinology

Article Title: CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization

doi: 10.1210/en.2018-01097

Figure Lengend Snippet: Study to characterize the promoting effects of CAMSAP2 KD on Sertoli cell TJ permeability function using the PFOS toxicant model. (A) Regimen used to characterize the promoting effects of CAMSAP2 KD on Sertoli cell TJ barrier function of n = 3 experiments. (B) Results of a representative experiment by quantifying changes in TER across the Sertoli cell epithelium on bicameral units to assess the TJ barrier function. CAMSAP2 KD promoted Sertoli cell TJ barrier function, whereas treatment of cells with PFOS (20 µM) perturbed the barrier function, consistent with earlier reports (112, 113). More importantly, CAMSAP2 KD was capable of blocking PFOS-mediated Sertoli cell TJ barrier disruption. Results of three additional experiments yielded similar results. *P < 0.05, by comparing between the PFOS and control RNAi groups by Student t test; **P < 0.01, by comparing between the PFOS and CAMSAP2 RNA plus PFOS groups. (C) IF analysis of corresponding BTB-associated proteins (green fluorescence). PFOS (20 µM) treatment perturbed distribution of TJ (e.g., CAR, ZO-1) and basal ES (e.g., N-cadherin, β-catenin). However, CAMSAP2 KD was capable of blocking PFOS-induced disruptive changes in TJ and basal ES protein distribution. Data shown are representative findings of an experiment from n = 3 independent experiments, which yielded similar results. Scale bar, 40 µm, which applies to all other micrographs. (D) IB analysis of a representative experiment illustrating that PFOS (20 µM) induced a downregulation of ZO-1 but no apparent effects on the steady-state levels of the TJ (CAR) and basal ES (N-cadherin, β-catenin) proteins, wherein GAPDH served as a protein loading control. Uncropped immunoblots are shown in an online repository (30). Bar graph on the right panel shows the composite data, with each bar representing a mean ± SD of n = 3 experiments. *P < 0.05; **P < 0.01.

Article Snippet: RT-PCR data reported herein are representative findings from an experiment of n = 3 experiments. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene Primer Sequence Position Length, bp Temp., °C Cycle No. GenBank Accession No. Camsap1 Sense: 5′-CTATCGGCGAGAACACCTCTC-3′ 1065–1085 166 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001168549.1","term_id":"270483893","term_text":"NM_001168549.1"}} NM_001168549.1 Antisense: 5′-CATCGATGGCACCTCCTTCA-3′ 1211–1230 Camsap2 Sense: 5′-CGCCTTGAAAGATGGGGGAT -3′ 665–684 172 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001134503.1","term_id":"197386167","term_text":"NM_001134503.1"}} NM_001134503.1 Antisense: 5′-CGAAGTTCCTCTGGCACAGT-3′ 817–836 Camsap3 Sense: 5′-GGTTACTCCTGGGTCCTGTCT-3′ 3970–3990 139 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001144840.1","term_id":"221325615","term_text":"NM_001144840.1"}} NM_001144840.1 Antisense: 5′-GGACCATGTCTCAGCTTCTCTTT-3′ 4086-4108 S16 Sense: 5′-TCCGCTGCAGTCCGTTCAAGTCTT-3′ 15–38 385 {"type":"entrez-nucleotide","attrs":{"text":"XM_341815","term_id":"293355792","term_text":"XM_341815"}} XM_341815 Antisense: 5′-GCCAAACTTCTTGGTTTCGCAGCG-3′ 376–399 Open in a separate window Primers Used for RT-PCR Experiments MT spin-down assay to assess the relative levels of polymerized MTs The MT spin-down assay was performed as earlier described ( 22 ) to estimate the relative levels of polymerized MTs vs free ( i.e. , nonpolymerized) tubulins in Sertoli cell lysates in treatment vs control groups using kits (catalog no. BK038) from Cytoskeleton (Denver, CO) and protocols from the manufacturer.

Techniques: Permeability, Blocking Assay, Disruption, Control, Fluorescence, Western Blot

Journal: Endocrinology

Article Title: CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization

doi: 10.1210/en.2018-01097

Figure Lengend Snippet: CAMSAP2 KD that blocks PFOS-mediated Sertoli cell TJ barrier disruption is mediated by its promoting effects on MT cytoskeletal organization through Akt1/2 signaling. Because CAMSAP2 is a −TIP and an MT regulator, we next used the regimen outlined in Fig. 4A to investigate the mechanism by which CAMSAP2 KD blocks PFOS-induced TJ barrier disruption through MT organization. (A) Study by IF microscopy confirmed the effective KD of CAMSAP2 in Sertoli cell epithelium, but PFOS had no apparent effects on the expression of CAMSAP2 in Sertoli cells (see also the enlarged images boxed in yellow in insets) as noted in the first column. Sertoli cells treated with PFOS (20 µM) alone were found to induce extensive cytoskeletal disorganization of MTs across the Sertoli cell cytosol, whereas MTs (visualized by staining of α-tubulin, which, together with β-tubulin, forms the α-/β-tubulin oligomers, which are the building blocks of MTs) were truncated (see enlarged image in inset boxed in yellow vs control and treatment groups), unlike MTs in the control (Ctrl RNAi) group, wherein MTs stretched across the Sertoli cell cytosol. Whereas MTs in cells of CAMSAP2 KD group appeared to have no differences from the control (Ctrl RNAi) group, CAMSAP2 KD was effective in blocking PFOS-induced Sertoli cell MT disorganization of MTs. EB1, a +TIP known to confer MT stabilization (108), appeared as small rodlike structures restrictively localized to the plus-ends of MTs across the Sertoli cell cytosol, consistent with data shown in Fig. 1D, as reported earlier (22). EB1 distribution in Sertoli cells following CAMSAP2 KD was not different from the control (Ctrl RNAi) group, and PFOS treatment caused maldistribution of EB1 along the MTs wherein EB1 no longer localized at the minus-ends of MTs. However, CAMSAP2 KD blocked the PFOS-induced mislocalization of EB1 in Sertoli cells. siGLO (red fluorescence) served as an indicator to confirm successful transfection. Scale bars, 40 µm (20 µm in the inset), which applies to corresponding micrographs and insets. (B) Lysates of Sertoli cells from the above experiments were used for IB analysis to investigate whether CAMSAP2 exerted its effects through signaling proteins recently shown to be involved in MT regulation or by modifying different molecular forms of MTs. It was noted that PFOS treatment led to a mild but consistent upregulation in CAMSAP2 expression, which could be blocked after CAMSAP2 KD (see also bar graph in the top right panel). Interestingly, the levels of EB1 [an MT stabilizer (108)] and MARK4 [an MT destabilizing protein kinase (116)] were unaffected. Also, the levels of the three molecular forms of MTs, including detyrosinated α-tubulin and acetylated α-tubulin [known to stabilize MTs (117–119)], as well as tyrosinated tubulin [known to promote MT dynamics (i.e., to destabilize MTs) (117)], were unaffected in all treatment groups vs control group. The same was true for the two phosphorylated/activated isoforms of rpS6, namely p-rpS6-S235/S236 and p-rpS6-S240/S244, whose activation (or upregulation) was known to induce Sertoli cell BTB remodeling (120). However, a mild but consistent downregulation of p-Akt1-S474 and p-Akt2-S474 was noted following PFOS treatment, wherein CAMSAP2 KD was no longer capable of altering PFOS-induced changes in such signaling protein expression. Uncropped immunoblots are shown in an online repository (30). Composite data are shown in the right panel, with each bar representing a mean ± SD of n = 3 experiments. *P < 0.05; **P < 0.01. ns, not significant.

Article Snippet: RT-PCR data reported herein are representative findings from an experiment of n = 3 experiments. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene Primer Sequence Position Length, bp Temp., °C Cycle No. GenBank Accession No. Camsap1 Sense: 5′-CTATCGGCGAGAACACCTCTC-3′ 1065–1085 166 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001168549.1","term_id":"270483893","term_text":"NM_001168549.1"}} NM_001168549.1 Antisense: 5′-CATCGATGGCACCTCCTTCA-3′ 1211–1230 Camsap2 Sense: 5′-CGCCTTGAAAGATGGGGGAT -3′ 665–684 172 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001134503.1","term_id":"197386167","term_text":"NM_001134503.1"}} NM_001134503.1 Antisense: 5′-CGAAGTTCCTCTGGCACAGT-3′ 817–836 Camsap3 Sense: 5′-GGTTACTCCTGGGTCCTGTCT-3′ 3970–3990 139 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001144840.1","term_id":"221325615","term_text":"NM_001144840.1"}} NM_001144840.1 Antisense: 5′-GGACCATGTCTCAGCTTCTCTTT-3′ 4086-4108 S16 Sense: 5′-TCCGCTGCAGTCCGTTCAAGTCTT-3′ 15–38 385 {"type":"entrez-nucleotide","attrs":{"text":"XM_341815","term_id":"293355792","term_text":"XM_341815"}} XM_341815 Antisense: 5′-GCCAAACTTCTTGGTTTCGCAGCG-3′ 376–399 Open in a separate window Primers Used for RT-PCR Experiments MT spin-down assay to assess the relative levels of polymerized MTs The MT spin-down assay was performed as earlier described ( 22 ) to estimate the relative levels of polymerized MTs vs free ( i.e. , nonpolymerized) tubulins in Sertoli cell lysates in treatment vs control groups using kits (catalog no. BK038) from Cytoskeleton (Denver, CO) and protocols from the manufacturer.

Techniques: Disruption, Microscopy, Expressing, Staining, Control, Blocking Assay, Fluorescence, Transfection, Activation Assay, Western Blot

Journal: Endocrinology

Article Title: CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization

doi: 10.1210/en.2018-01097

Figure Lengend Snippet: CAMSAP2 KD that blocks PFOS-mediated Sertoli cell TJ barrier disruption is mediated by its promoting effects on MT dynamics. (A) Biochemical-based MT spin-down assay that assessed the level of polymerized MTs using lysates of Sertoli cells from the three treatment groups vs the control (Ctrl RNAi) group based on the regimen in Fig. 4A. Uncropped blots are shown in an online repository (30). The left panel shows results of a representative experiment from n = 3 independent experiments that yielded similar results, and the composite data are shown in the right panel. Each bar represents a mean ± SD of n = 3 experiments, and each sample had duplicate cultures. *P < 0.05; **P < 0.01. (B) Biochemical MT polymerization kinetics assays. Findings shown in the left panel are representative data of an experiment from n = 3 experiments that yielded similar results. Sertoli cell lysates were used for a fluorometric-based MT polymerization assay as detailed in “Materials and Methods,” in which the incorporation of a fluorescent reporter into MTs during polymerization was monitored by enhanced fluorescence emission at 430 nm (excitation wavelength, 360 nm) in the three treatment groups vs the control (Ctrl RNAi) group, including the positive and negative control groups as shown. The rate of MT polymerization that measured an increase in fluorescence intensity over time during the initial linear phase in the first 10 min as noted in the left panel was estimated by linear regression, and the results are shown in the right panel. Each bar in the histogram in the right panel represents a mean ± SD of n = 3 independent experiments. The rate of MT polymerization in the control (Ctrl RNAi) was arbitrarily set at 1. *P < 0.05, **P < 0.01, by Student t test for paired comparisons.

Article Snippet: RT-PCR data reported herein are representative findings from an experiment of n = 3 experiments. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene Primer Sequence Position Length, bp Temp., °C Cycle No. GenBank Accession No. Camsap1 Sense: 5′-CTATCGGCGAGAACACCTCTC-3′ 1065–1085 166 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001168549.1","term_id":"270483893","term_text":"NM_001168549.1"}} NM_001168549.1 Antisense: 5′-CATCGATGGCACCTCCTTCA-3′ 1211–1230 Camsap2 Sense: 5′-CGCCTTGAAAGATGGGGGAT -3′ 665–684 172 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001134503.1","term_id":"197386167","term_text":"NM_001134503.1"}} NM_001134503.1 Antisense: 5′-CGAAGTTCCTCTGGCACAGT-3′ 817–836 Camsap3 Sense: 5′-GGTTACTCCTGGGTCCTGTCT-3′ 3970–3990 139 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001144840.1","term_id":"221325615","term_text":"NM_001144840.1"}} NM_001144840.1 Antisense: 5′-GGACCATGTCTCAGCTTCTCTTT-3′ 4086-4108 S16 Sense: 5′-TCCGCTGCAGTCCGTTCAAGTCTT-3′ 15–38 385 {"type":"entrez-nucleotide","attrs":{"text":"XM_341815","term_id":"293355792","term_text":"XM_341815"}} XM_341815 Antisense: 5′-GCCAAACTTCTTGGTTTCGCAGCG-3′ 376–399 Open in a separate window Primers Used for RT-PCR Experiments MT spin-down assay to assess the relative levels of polymerized MTs The MT spin-down assay was performed as earlier described ( 22 ) to estimate the relative levels of polymerized MTs vs free ( i.e. , nonpolymerized) tubulins in Sertoli cell lysates in treatment vs control groups using kits (catalog no. BK038) from Cytoskeleton (Denver, CO) and protocols from the manufacturer.

Techniques: Disruption, Spin Down Assay, Control, Polymerization Assay, Fluorescence, Negative Control

Journal: Endocrinology

Article Title: CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization

doi: 10.1210/en.2018-01097

Figure Lengend Snippet: CAMSAP2 KD that blocks PFOS-mediated Sertoli cell TJ barrier disruption is mediated by its promoting effects on actin cytoskeletal organization and actin dynamics. The regimen shown in Fig. 4A was used to examine the promoting effects of CAMSAP2 KD on F-actin organization and function. (A) In Sertoli cells of control cell cultures treated with nontargeting negative control siRNA duplexes (Ctrl RNAi) (Table 1), actin filaments (green fluorescence) stretched across the cell cytosol to support cell shape and other physiological functions (e.g., endosome-mediated trafficking). PFOS (20 µM) treatment caused disorganization of actin filaments wherein these filaments were truncated and no longer stretched across the cell cytosol. CAMSAP2 KD, however, was capable of blocking PFOS-induced Sertoli cell injury regarding actin filament organization (see also enlarged images in insets from corresponding yellow boxed areas for better visualization), but also the proper distribution of the actin regulatory proteins (green fluorescence) as noted in the CAMSAP2 RNAi plus PFOS group, necessary to confer actin dynamics to support Sertoli cell TJ barrier function. These include Arp3 [an actin branched polymerization protein (123)] and Eps8 [an actin barbed-end capping and bundling protein (119, 124)]. Overexpression of CAMSAP2 was able to block PFOS-induced maldistribution of these actin regulatory proteins. Scale bars, 40 µm; 20 µm in insets, the magnified image of the yellow boxed area. Sertoli cell nuclei were visualized by DAPI (blue). siGLO (red fluorescence) served as an indicator for successful transfection. (B) Steady-state protein levels of Arp3 and Eps8 in different treatment groups vs the control group, wherein β-actin served as a protein loading control. These are representative findings from an experiment of n = 3 experiments that yielded similar observations. Uncropped immunoblots are shown in an online repository (30). (C) In this biochemical-based actin bundling assay, the ability of Sertoli cell lysate to bundle actin filaments in treatment vs control (Ctrl RNAi) groups was compared. Uncropped blots are shown in an online repository (30). It was noted that CAMSAP2 KD was able to induce (vs PFOS, which was shown to reduce) actin bundling activity but was also capable of blocking PFOS-induced decline in actin bundling activity. GAPDH served as the protein loading control. Lysates from this experiment used to probe for CAMSAP2 illustrate the successful KD of CAMSAP2. Findings in the left panel show a representative spin-down assay of n = 3 experiments. Histogram in the right panel shows the composite data in which each bar represents a mean ± SD of n = 3 experiments. *P < 0.05; **P < 0.01. (D) Sertoli cells (0.4 × 106 cells/cm2) cultured in vitro were used for this assay in which lysates obtained from cells of different treatment vs control groups were used for a fluorometric-based actin polymerization assay, in which the polymerization of pyrene-labeled actin was monitored by enhanced fluorescence emission at 395 to 440 nm, as noted in a representative experiment (left panel). The kinetics of actin polymerization were assessed by quantifying increases in fluorescence intensity over time during the initial linear phase in the first 10 min by linear regression analysis. The histogram in the right panel shows the composite data of the kinetics analysis, wherein each bar represents a mean ± SD of n = 3 independent experiments with triplicate cultures in each experiment. The rate of actin polymerization in Ctrl RNAi control group was arbitrarily set at 1, against which statistical analysis was performed. *P < 0.05; **P < 0.01.

Article Snippet: RT-PCR data reported herein are representative findings from an experiment of n = 3 experiments. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene Primer Sequence Position Length, bp Temp., °C Cycle No. GenBank Accession No. Camsap1 Sense: 5′-CTATCGGCGAGAACACCTCTC-3′ 1065–1085 166 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001168549.1","term_id":"270483893","term_text":"NM_001168549.1"}} NM_001168549.1 Antisense: 5′-CATCGATGGCACCTCCTTCA-3′ 1211–1230 Camsap2 Sense: 5′-CGCCTTGAAAGATGGGGGAT -3′ 665–684 172 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001134503.1","term_id":"197386167","term_text":"NM_001134503.1"}} NM_001134503.1 Antisense: 5′-CGAAGTTCCTCTGGCACAGT-3′ 817–836 Camsap3 Sense: 5′-GGTTACTCCTGGGTCCTGTCT-3′ 3970–3990 139 60 35 {"type":"entrez-nucleotide","attrs":{"text":"NM_001144840.1","term_id":"221325615","term_text":"NM_001144840.1"}} NM_001144840.1 Antisense: 5′-GGACCATGTCTCAGCTTCTCTTT-3′ 4086-4108 S16 Sense: 5′-TCCGCTGCAGTCCGTTCAAGTCTT-3′ 15–38 385 {"type":"entrez-nucleotide","attrs":{"text":"XM_341815","term_id":"293355792","term_text":"XM_341815"}} XM_341815 Antisense: 5′-GCCAAACTTCTTGGTTTCGCAGCG-3′ 376–399 Open in a separate window Primers Used for RT-PCR Experiments MT spin-down assay to assess the relative levels of polymerized MTs The MT spin-down assay was performed as earlier described ( 22 ) to estimate the relative levels of polymerized MTs vs free ( i.e. , nonpolymerized) tubulins in Sertoli cell lysates in treatment vs control groups using kits (catalog no. BK038) from Cytoskeleton (Denver, CO) and protocols from the manufacturer.

Techniques: Disruption, Control, Negative Control, Fluorescence, Blocking Assay, Over Expression, Transfection, Western Blot, Activity Assay, Spin Down Assay, Cell Culture, In Vitro, Polymerization Assay, Labeling

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test